Four-region µCT analysis detects compensatory ventilation in the right caudal lobe of a mouse fibrosis model that whole-lung readouts miss
Synopsis
In 22 C57BL/6 mice (11 given triple oropharyngeal bleomycin at 0.25 mg/kg to induce pulmonary fibrosis and 11 given saline), respiratory-gated µCT was acquired at baseline and days 7, 14, and 21, a U-Net deep-learning algorithm segmented the lungs into left and right lobes that were further divided into apical and caudal regions using airway landmarks for four subregions, and regional volumes, aeration compartments, and ventilation maps were extracted; saline-treated mice showed stable metrics with minimal interanimal variability, whereas bleomycin-treated animals showed early and heterogeneous fibrotic changes with the apical regions most affected and the caudal-right region displaying a compensatory functional increase, so that µCT-based regional analysis detected localized dysfunction a
Micro-computed tomography–guided regional lung segmentation and representative quantitative outputs. (A) Manual identification of anatomical landmarks used to define lung subregions viewed in the coronal plane of a representative bleomycin (BLM) animal: Red spheres indicate the first and second bifurcations of the left main bronchus and the origin of the right accessory lobe. Red dashed lines represent the apico-caudal division thresholds (Threshold Left, ThL and Threshold Right, ThR), defined based on these landmarks; black dashed lines indicate the measured distance between bifurcations on the left bronchus. Airways are represented with a 3D green rendering (B) example of the resulting lung segmentation mask into 4 subregions in the coronal plane: apical-left (Api L), caudal-left (Cau L), apical-right (Api R), and caudal-right (Cau R). (C, D) Voxel-wise histograms of lung attenuation (Hounsfield units [HU]) and specific gas volume change (DSVg, mL/g) for each subregion in a representative BLM case at day 21, illustrating regional differences in lung structure and ventilation.
PubMedInterpretation
The study establishes and validates a µCT-based four-subregion (apical-left, caudal-left, apical-right, caudal-right) longitudinal pipeline that yields both structural (volume, mean lung attenuation, aeration compartments) and functional (tidal volume, median ΔSVg, ventilation compartments) biomarkers. Earlier regional µCT work was either restricted to small volumes of interest around bronchial bifurcations without functional readouts, or segmented only left and right lungs and could miss lobar compensation, or used arbitrary apical-caudal thresholds that limit longitudinal robustness; this study defines anatomical boundaries from airway landmarks (first and second bifurcations of the left main bronchus and the origin of the accessory lobe on the right) and couples regional segmentation with ventilation maps. In 11 saline-treated control mice, whole-lung volume remained constant at approximately 600 mm³, normo-aerated tissue accounted for approximately 80% of total lung at end expiration (68%-83% across subregions), and subregional tidal volumes were stable and followed the same anatomical pattern as static lung volumes, with minimal error bars supporting reproducibility and physiological consistency.
Bleomycin-induced fibrosis was spatially heterogeneous, with apical regions, particularly apical-left, affected earliest and most severely, while caudal regions followed distinct temporal dynamics. Whole-lung volume transiently increased at day 7 (P < .001), driven by expansion in caudal-right and apical-right, while apical-left decreased significantly from day 14 onward (P = .0052); normo-aerated tissue in apical-left dropped to approximately 30% by day 7 and to approximately 20% by day 14 (P < .001), and its normal ventilation declined significantly already at day 7 (P = .0097), reaching approximately 19% by day 21. Longitudinal µCT was acquired at baseline and days 7, 14, and 21, with one- or two-way ANOVA and Dunnett or Tukey post hoc tests and a significance threshold of P < .05, reporting fold changes relative to baseline across multiple subregional parameters.
The right caudal lobe showed functional compensation despite persistent structural damage: ventilation transiently declined at day 14 and recovered to baseline by day 21, while residual non-aerated and non-ventilated voxels persisted. This structure-function mismatch points to regional compensatory mechanisms rather than spontaneous resolution of fibrosis, aligning with prior reports of functional compensation in spared lung regions after injury or lobectomy; whole-lung endpoints cannot distinguish such local compensation from genuine improvement. In caudal-right, %GasP02 modestly increased at day 7 (P = .0473), ventilation transiently declined (tidal volume P = .0407, ΔSVg P < .001, %Low-Vent P = .0504), ventilation impairment peaked at day 14 (%Normal-Vent P = .0387, %Non-Vent P = .00720), and function recovered by day 21 although residual non-aerated and non-ventilated voxels remained significant (P < .05).
The left caudal region showed functional impairment preceding structural remodeling, suggesting early subclinical dysfunction. Unlike apical regions where structural and functional deterioration tracked together, caudal-left showed early reductions in tidal volume and ΔSVg (P < .001) and increased %Low-Vent (P = .0349), while structural remodeling only became evident by day 14 and worsened through day 21, with overall damage still less severe than in apical regions. Based on longitudinal regional µCT data from the same cohort, with parameters expressed as fold changes relative to baseline and corresponding P values reported.
Perspective
The pipeline is intended for preclinical idiopathic pulmonary fibrosis research and for settings that require longitudinal tracking of regional structure-function changes, such as efficacy assessment and treatment-window exploration for antifibrotic drugs; the authors consider it applicable to other pulmonary disease models characterized by regional heterogeneity. For readers, this means regional ventilation maps can serve as complementary evidence when interpreting whole-lung functional readouts in the bleomycin model, helping to identify localized improvement or deterioration masked by global metrics.
Histological validation cannot be performed at the level of the four in vivo µCT subregions because fixation, embedding, and sectioning alter lung geometry, leaving only left-right comparisons reliable; the authors cite prior work showing side-level correlations between µCT biomarkers and histological fibrosis scores. The pipeline relies on automated left-right lung segmentation, which may not be readily implementable in all laboratories due to computational or technical constraints; airway segmentation remains manual or semi-automatic and is a bottleneck for full automation and scalability; and the four-region division captures major heterogeneity, while finer segmentation could improve sensitivity to early changes. In addition, the mouse model shows apical-predominant involvement, which differs from human idiopathic pulmonary fibrosis, so the value of regional analysis for interpreting compensatory responses and structure-function relationships still needs testing in more models.