Longitudinal microcomputed tomography uncovers divergent trajectories of lung maturation in preterm and hyperoxia-exposed neonatal rabbits

Preterm birth disrupts critical stages of lung development, including alveolarization and microvascular maturation, and is a major contributor to long-term respiratory morbidity. Understanding how lung structure and function evolve during the early postnatal period is essential to elucidate the developmental origins of chronic lung disease.^1,2 This is particularly relevant in bronchopulmonary dysplasia (BPD), a chronic respiratory disorder of extremely premature infants (<28 weeks gestational age) characterized by arrested alveolarization, simplified distal airspaces, dysmorphic microvasculature, and persistent inflammation or fibrosis, largely induced by perinatal insults such as mechanical ventilation, hyperoxia, and infections.^3-5

Animal models are essential to advance our understanding of these processes. Prematurely delivered rabbits exposed to high oxygen concentrations reproduce key features of human neonatal lung immaturity and BPD, including impaired alveolarization, abnormal vascular development, and vulnerability to oxygen injury. Catozzi et al. established and histologically characterized a rabbit model of BPD,^6,7 reporting impaired lung maturation observed as a reduction in radial alveolar count (RAC) and simplified distal airspaces in premature subjects compared with term controls, with further impairment under hyperoxia. These histological alterations are expected to limit lung aeration and ventilation.

This model provides a translational platform to investigate disrupted lung growth in early life. However, longitudinal, noninvasive tools capable of simultaneously assessing structural and functional changes during lung maturation remain lacking.

In this study, we introduce respiratory-gated microcomputed tomography (μCT) as a noninvasive method to longitudinally quantify, in the previously characterized BPD model, structural and functional biomarkers of neonatal lung development.

Time-mated male and female New Zealand White rabbits were obtained from Charles River Laboratories (Domaine des Oncins, France) and maintained under controlled environmental conditions (15-21°C, 55% ± 15% relative humidity, 12:12 h light/dark cycle) with ad libitum access to food and water. New Zealand White rabbits were delivered spontaneously at term (gestational day 31, term, n = 8) or preterm by cesarean section (day 28, preterm, n = 16). Term rabbits were exposed to normoxia (NOX; 21% O₂), while preterm rabbits were exposed to either normoxia (21% O₂, n = 8) or hyperoxia (HyOX; 70% O₂, n = 8). Respiratory-gated μCT acquired in free-breathing condition was performed longitudinally at early time point (t1: postnatal day 4, term; day 7, preterm) and at late time point (t2: day 10, term; day 13, preterm), enabling reconstruction of end-inspiratory and end-expiratory volumes at 90-μm isotropic voxel resolution.^8,9 Two morphofunctional biomarkers were derived: specific gas volume at end-expiration (SVg_EXP, ml/g), reflecting structural aeration and alveolar growth, and change in specific gas volume (ΔSVg = SVgI_NSP − SVg_EXP, ml/g), reflecting ventilation dynamics and functional adaptation. This dual-parameter framework provides an integrated readout of lung maturation, distinguishing structural expansion from compensatory ventilatory adjustments.^10,11 A subset of 13 rabbits was sacrificed at terminal time points for hematoxylin and eosin (H&E) staining and RAC calculation, all performed according to the protocols detailed by Catozzi  et al.⁶

Representative color-coded μCT lung maps (Figure 1B) show SVg_EXP and ΔSVg distributions in term, preterm NOX, and preterm HyOX rabbits. At t1, all groups displayed comparable SVg_EXP (term: 0.43 ± 0.05; preterm NOX: 0.42 ± 0.04; preterm HyOX: 0.42 ± 0.06) and ΔSVg (term: 0.14 ± 0.01; preterm NOX: 0.15 ± 0.01; preterm HyOX: 0.11 ± 0.01), indicating similar early aeration and ventilation. By t2, however, distinct trajectories emerged: term rabbits showed a marked increase in SVg_EXP with stable ΔSVg values (SVg_EXP: 0.67 ± 0.05; ΔSVg: 0.16 ± 0.01), consistent with normal alveolar expansion and maturation. Preterm NOX rabbits, instead, exhibited a modest SVg_EXP increase (0.52 ± 0.04) but a substantial rise in ΔSVg (0.24 ± 0.01), suggesting functional compensation for delayed structural growth. In contrast, preterm HyOX rabbits showed reduced SVg_EXP (0.30 ± 0.04) and a limited ΔSVg increase (0.14 ± 0.01), indicating oxygen-induced impairment of both structural and functional lung maturation. Consistent with these findings, representative H&E stains at t2 (Figure 1B) revealed physiologically developed lungs in term animals (RAC: 9.51 ± 1.53), whereas preterm NOX rabbits exhibited premature lungs with simplified alveoli (RAC: 7.74 ± 0.63), and preterm HyOX subjects displayed simplified distal airspaces, inflammatory recruitment, and disrupted alveoli (RAC: 7.01 ± 1.24). Furthermore, the Spearman correlation calculated between RAC and SVg_EXP at t2 revealed a significant positive correlation (R = 0.80, P < .05), confirming a robust interconnection between lung aeration and alveolar count.

Figure 1.

Longitudinal assessment of lung maturation in term and preterm rabbits by respiratory-gated μCT. (A) Experimental design: term rabbits (n = 8) were delivered spontaneously at gestational day 31; preterm rabbits (n = 16) were delivered at day 28 via cesarean section and exposed to either normoxia (NOX, 21% O₂; n = 8) or hyperoxia (HyOX, 70% O₂; n = 8; preterm animals only). All rabbits underwent μCT at early time point (t1): postnatal days 4(term)/7(preterm) and late time point (t2): 10(term)/13(preterm). (B) Representative μCT maps: Color-coded μCT images show specific gas volume at end-expiration (SVg_EXP, ml/g) and ventilatory change (ΔSVg, ml/g) in term NOX, preterm NOX, and preterm HyOX rabbits at t1 and t2. Reported values represent the range (min-max) across all conditions. At t1, term rabbits showed SVg_EXP 0.235-0.620 and ΔSVg 0.125-0.188; preterm NOX rabbits showed SVg_EXP 0.270-0.638 and ΔSVg 0.113-0.212; preterm HyOX rabbits showed SVg_EXP 0.218-0.638 and ΔSVg 0.075-0.156. At t2, term rabbits showed SVg_EXP 0.515-0.891 and ΔSVg 0.119-0.225; preterm NOX rabbits showed SVg_EXP 0.375-0.690 and ΔSVg 0.188-0.294; preterm HyOX rabbits showed SVg_EXP 0.165-0.462 and ΔSVg 0.088-0.206. (B') Representative images of hematoxylin and eosin–stained histological sections from term, preterm NOX, and preterm HyOX mice at t2, magnified at 20x; bar length is 100 μm. (C-E) Integrated morphofunctional analysis: ΔSVg was plotted against SVg_EXP for individual animals (light arrows) and group averages (dark arrows) between t1 and t2. Slopes (m) represent the balance between structural and functional maturation. Term rabbits showed a balanced growth (m = 0.05; C), preterm NOX rabbits displayed delayed alveolarization with compensatory ventilation (m = 0.89; D), and preterm HyOX rabbits exhibited a regression in aeration with impaired functional development (m = –0.31; E). Abbreviations: ΔSVg, change in specific gas volume; SVg_EXP, specific gas volume at end-expiration; μCT, microcomputed tomography; HyOX, hyperoxia; NOX, normoxia.

To quantitatively represent the developmental trajectories, we plotted ΔSVg against SVg_EXP between t1 and t2 for each animal (light arrows) and group averages (dark arrows) (Figure 1C-E). The slope of this trajectory (designated as m) serves as an integrated morphofunctional biomarker of maturation. Statistical differences were tested using multivariate analysis of variance. Term rabbits exhibited a nearly flat trajectory (m = 0.05; Figure 1C), indicating balanced alveolar growth with stable ventilation. Preterm NOX rabbits displayed a significantly steeper slope (m = 0.89, P < .001 vs term; Figure 1D), reflecting disproportionate ventilatory adaptation to immature structure. Preterm HyOX rabbits exhibited a negative slope (m = –0.31, P < .001 vs term; P < .01 vs preterm NOX; Figure 1E), consistent with regression in aeration and impaired functional development.

This integrated analysis, corroborated by independent histological readouts, revealed strikingly divergent developmental trajectories—normal maturation, compensatory adaptation, and oxygen-induced impairment—that would not have been detected using only structural or functional metrics. Longitudinal μCT thus provided mechanistic insight into how prematurity and oxygen exposure shape neonatal lung development, distinguishing adaptive responses from pathological regression. Specifically, μCT differentiated normal lung maturation in term rabbits from delayed development in preterm NOX rabbits, which nevertheless exhibited functional compensation over time, and identified both reduced aeration and limited functional progression in preterm HyOX rabbits, highlighting the detrimental impact of sustained hyperoxia. Additional μCT-derived parameters (lung volume, lung density, tidal volume, and breathing rate) showed trends consistent with the morphofunctional pattern reported here (data not shown), in line with previous μCT findings.⁶

Despite the clear group differences, the relatively small sample size represents a limitation requiring consideration in future validation studies. Classical pulmonary function tests should also be evaluated as potentially informative complements, and benchmarks, to μCT, although their terminal nature precludes longitudinal follow-up. Future research should include earlier μCT acquisitions to better characterize the influence of prematurity on developmental trajectories, link μCT biomarkers to multiple molecular and histological endpoints to uncover the underlying cellular mechanisms, and assess potential sex-specific effects.

In conclusion, we provide here a proof-of-concept demonstration of the potential of respiratory-gated μCT as a powerful longitudinal tool to noninvasively monitor neonatal lung maturation in a very early postnatal window, integrating structural and functional biomarkers within a single readout. This approach revealed 3 divergent developmental trajectories in term, preterm, and hyperoxia-exposed rabbits, highlighting both adaptive and pathological pathways of lung growth. Altogether, the present findings point to μCT as a translationally relevant tool to study the balance between alveolar growth and ventilatory adaptation, with direct implications for a better understanding of the origins of chronic lung disease following preterm birth.

Author contributions

D.B., F.P., and F.F.S. conceived and designed research. E.F., G.A., and C.C. performed in vivo experiments. D.B. and F.P. analyzed and/or interpreted data. D.B. and M.B. prepared figures. D.B., F.P., E.F., and F.F.S. drafted the manuscript. M.B., G.A., G.V., C.C., and A.A. edited and revised the manuscript. All authors read and approved the final manuscript.

Supplementary material

Supplementary material is available at American Journal of Respiratory Cell and Molecular Biology online.

Conflicts of interest

F.F.S., E.F., G.A., G.V., and C.C. are employees of Chiesi Farmaceutici S.p.A. that supported the research work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be perceived as a potential conflict of interest. Please see the ICMJE disclosure forms, which have been provided as supplementary material.

Funding

This study was fully supported by Chiesi Farmaceutici S.p.A. and by the National Plan for NRRP Complementary Investments (PNC, established by decree-law of May 6, 2021, no. 59, converted into law no. 101 of 2021), within the call for funding of research initiatives on technologies and innovative trajectories in the health and care sectors (Directorial Decree no. 931 of 06-06-2022)—project no. PNC0000003—AdvaNced Technologies for Human-centrEd Medicine (project acronym: ANTHEM).

Data availability

All data presented in this manuscript are available from the authors for noncommercial use, subject to reasonable request and completion of the required data access agreements. The original code used for the analysis of μCT imaging data can be obtained from the lead contact upon request. Any additional information necessary to reanalyze the data reported in this study is also available from the lead contact.