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Micro-computed tomography (micro-CT) imaging is an emerging technology with many applications in small animals, for example, the study of pulmonary diseases, although clear guidelines and critical mass of evidence are still missing in the preclinical literature. The neonatal rabbit is a valuable model for studying pulmonary development. However, the longitudinal monitoring of lung function by micro-CT can be challenging. Distinctive datasets corresponding to the end-inspiration and end-expiration phases need to be generated and analyzed to derive lung-functional parameters. The quality of CT scans and the reliability of parameters obtained remain highly dependent on the anesthesia protocol used. Three different anesthetic protocols were tested. The combination of dexmedetomidine 0.25 mg/kg injected intraperitoneally followed by 1% isoflurane was found to facilitate CT imaging at 4 and 11 days after birth. Contrarily, isoflurane and ketamine-xylazine were found unsuitable and thus not investigated further. Total lung volumes significantly increased at day 11 compared with baseline in both respiratory phases, whereas lung tissue remained constant. As expected, functional residual capacity, air-to-tissue ratio, and minute ventilation were significantly increased at day 11 in each animal. Those parameters were correlated with inspiratory capacity, compliance, elastance, and resistance of both respiratory system and tissue component, as measured by flexiVent. Lung development was also evaluated by histomorphometric analyses. In conclusion, we have identified a safe and suitable anesthesia protocol for micro-CT imaging in neonatal rabbits. Moreover, the possibility to longitudinally measure lung function in the same subject dramatically reduced the intraexperimental variability.

Bronchopulmonary dysplasia (BPD) is a complex condition frequently occurring in preterm newborns, and different animal models are currently used to mimic the pathophysiology of BPD. The comparability of animal models depends on the availability of quantitative data obtained by minimally biased methods. Therefore, the aim of this study was to provide the first design-based stereological analysis of the lungs in the hyperoxia-based model of BPD in the preterm rabbit. Rabbit pups were obtained on gestation day 28 (three days before term) by cesarean section and exposed to normoxic (21% O2, n = 8 ) or hyperoxic (95% O2, n = 8 ) conditions. After seven days of exposure, lung function testing was performed, and lungs were taken for stereological analysis. In addition, the ratio between pulmonary arterial acceleration and ejection time (PAAT/PAET) was measured. Inspiratory capacity and static compliance were reduced whereas tissue elastance and resistance were increased in hyperoxic animals compared with normoxic controls. Hyperoxic animals showed signs of pulmonary hypertension indicated by the decreased PAAT/PAET ratio. In hyperoxic animals, the number of alveoli and the alveolar surface area were reduced by one-third or by approximately 50% of control values, respectively. However, neither the mean linear intercept length nor the mean alveolar volume was significantly different between both groups. Hyperoxic pups had thickened alveolar septa and intra-alveolar accumulation of edema fluid and inflammatory cells. Nonparenchymal blood vessels had thickened walls, enlarged perivascular space, and smaller lumen in hyperoxic rabbits in comparison with normoxic ones. In conclusion, the findings are in line with the pathological features of human BPD. The stereological data may serve as a reference to compare this model with BPD models in other species or future therapeutic interventions.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm delivery, with significant morbidity and mortality in a neonatal intensive care setting. Research in this field aims to identify the mechanisms of late lung development with possible therapeutic targets and the improvement of medical management. Rabbits represent a suitable lab preclinical tool for mimicking the clinical BPD phenotype. Rabbits are born at term in the alveolar phase as occurs in large animals and humans and in addition, they can be delivered prematurely in contrast to mice and rats. Continuous exposure to high oxygen concentration (95% O

Evolving bronchopulmonary dysplasia (BPD) is characterized by impaired alveolarization leading to lung aeration inhomogeneities. Hyperoxia-exposed preterm rabbits have been proposed to mimic evolving BPD; therefore, we aimed to verify if this model has the same lung ultrasound and mechanical features of evolving BPD in human neonates. Semiquantitative lung ultrasound and lung mechanics measurement was performed in 25 preterm rabbits (28 days of gestation) and 25 neonates (mean gestational age ≈ 26 wk) with evolving BPD. A modified rabbit lung ultrasound score (rLUS) and a validated neonatal lung ultrasound score (LUS) were used. Lung ultrasound images were recorded and evaluated by two independent observers blinded to each other's evaluation. Lung ultrasound findings were equally heterogeneous both in rabbits as in human neonates and encompassed all the classical lung ultrasound semiology. Lung ultrasound and histology examination were also performed in 13 term rabbits kept under normoxia as further control and showed the absence of ultrasound and histology abnormalities compared with hyperoxia-exposed preterm rabbits. The interrater absolute agreement for the evaluation of lung ultrasound images in rabbits was very high [ICC: 0.989 (95%CI: 0.975-0.995)

Graphical abstract Corticosteroids as budesonide can be effective in reducing topic inflammation processes in different organs. Therapeutic use of budesonide in respiratory diseases, like asthma, chronic obstructive pulmonary disease, and allergic rhinitis is well known. However, the pulmonary distribution of budesonide is not well understood, mainly due to the difficulties in tracing the molecule in lung samples without the addition of a label. In this paper, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging protocol that can be used to visualize the pulmonary distribution of budesonide administered to a surfactant-depleted adult rabbit. Considering that budesonide is not easily ionized by MALDI, we developed an on-tissue derivatization method with Girard’s reagent P followed by ferulic acid deposition as MALDI matrix. Interestingly, this sample preparation protocol results as a very effective strategy to raise the sensitivity towards not only budesonide but also other corticosteroids, allowing us to track its distribution and quantify the drug inside lung samples. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03393-6.

Background In preterm infants, InSurE (Intubation-Surfactant-Extubation) and LISA (less invasive surfactant administration) techniques allow for exogenous surfactant administration while reducing lung injury associated with mechanical ventilation. We compared the acute pulmonary response and lung deposition of surfactant by LISA and InSurE in surfactant-depleted adult rabbits. Methods Twenty-six spontaneously breathing surfactant-depleted adult rabbits (6-7 weeks old) with moderate RDS and managed with nasal continuous positive airway pressure were randomized to 3 groups: (1) 200 mg/kg of surfactant by InSurE; (2) 200 mg/kg of surfactant by LISA; (3) no surfactant treatment (Control). Gas exchange and lung mechanics were monitored for 180 min. After that, surfactant lung deposition and distribution were evaluated monitoring disaturated-phosphatidylcholine (DSPC) and surfactant protein C (SP-C), respectively. Results No signs of recovery were found in the untreated animals. After InSurE, oxygenation improved more rapidly compared to LISA. However, at 180' LISA and InSurE showed comparable outcomes in terms of gas exchange, ventilation parameters, and lung mechanics. Neither DSPC in the alveolar pool nor SP-C signal distributions in a frontal lung section were significantly different between InSurE and LISA groups. Conclusions In an acute setting, LISA demonstrated efficacy and surfactant lung delivery similar to that of InSurE in surfactant-depleted adult rabbits. Impact Although LISA technique is gaining popularity, there are still several questions to address. This is the first study comparing LISA and InSurE in terms of gas exchange, ventilation parameters, and lung mechanics as well as surfactant deposition and distribution. In our animal study, three hours post-treatment, LISA method seems to be as effective as InSurE and showed similar surfactant lung delivery. Our findings provide some clarifications on a fair comparison between LISA and InSurE techniques, particularly in terms of surfactant delivery. They should reassure some of the concerns raised by the clinical community on LISA adoption in neonatal units.

BackgroundThe intratracheal (IT) administration of budesonide using surfactant as a vehicle has been shown to reduce the incidence of bronchopulmonary dysplasia (BPD) in preterm infants. The objective of this study was to characterize the in vitro characteristics and in vivo safety and efficacy of the extemporaneous combination of budesonide and poractant alfa.MethodsThe stability, minimum surface tension, and viscosity of the preparation were evaluated by means of high-performance liquid chromatography (HPLC), Wilhelmy balance, and Rheometer, respectively. The safety and efficacy of the IT administration of the mixture were tested in two respiratory distress syndrome (RDS) animal models: twenty-seventh day gestational age premature rabbits and surfactant-depleted adult rabbits.ResultsA pre-formulation trial identified a suitable procedure to ensure the homogeneity and stability of the formulation. Wilhelmy Balance tests clarified that budesonide supplementation has no detrimental effect on poractant alfa surface tension activity. The addition of budesonide to poractant alfa did not affect the physiological response to surfactant treatment in both RDS animal models, and was associated to a significant reduction of lung inflammation in surfactant-depleted rabbits.ConclusionOur in vitro and in vivo analysis suggests that the IT administration of a characterized extemporaneous combination of poractant alfa and budesonide is a safe and efficacious procedure in the context of RDS.

Background Non-invasive delivery of nebulized surfactant has been a neonatology long-pursued goal. Nevertheless, the clinical efficacy of nebulized surfactant remains inconclusive, in part, due to the great technical challenges of depositing nebulized drugs in the lungs of preterm infants. The aim of this study was to investigate the feasibility of delivering nebulized surfactant (poractant alfa) in vitro and in vivo with an adapted, neonate-tailored aerosol delivery strategy. Methods Particle size distribution of undiluted poractant alfa aerosols generated by a customized eFlow-Neos nebulizer system was determined by laser diffraction. The theoretical nebulized surfactant lung dose was estimated in vitro in a clinical setting replica including a neonatal continuous positive airway pressure (CPAP) circuit, a cast of the upper airways of a preterm neonate, and a breath simulator programmed with the tidal breathing pattern of an infant with mild respiratory distress syndrome (RDS). A dose-response study with nebulized surfactant covering the 100–600 mg/kg nominal dose-range was conducted in RDS-modelling, lung-lavaged spontaneously-breathing rabbits managed with nasal CPAP. The effects of nebulized poractant alfa on arterial gas exchange and lung mechanics were assessed. Exogenous alveolar disaturated-phosphatidylcholine (DSPC) in the lungs was measured as a proxy of surfactant deposition efficacy. Results Laser diffraction studies demonstrated suitable aerosol characteristics for inhalation (mass median diameter, MMD = 3 μm). The mean surfactant lung dose determined in vitro was 13.7% ± 4.0 of the 200 mg/kg nominal dose. Nebulized surfactant delivered to spontaneously-breathing rabbits during nasal CPAP significantly improved arterial oxygenation compared to animals receiving CPAP only. Particularly, the groups of animals treated with 200 mg/kg and 400 mg/kg of nebulized poractant alfa achieved an equivalent pulmonary response in terms of oxygenation and lung mechanics as the group of animals treated with instilled surfactant (200 mg/kg). Conclusions The customized eFlow-Neos vibrating-membrane nebulizer system efficiently generated respirable aerosols of undiluted poractant alfa. Nebulized surfactant delivered at doses of 200 mg/kg and 400 mg/kg elicited a pulmonary response equivalent to that observed after treatment with an intratracheal surfactant bolus of 200 mg/kg. This bench-characterized nebulized surfactant delivery strategy is now under evaluation in Phase II clinical trial (EUDRACT No.:2016–004547-36). Electronic supplementary material The online version of this article (10.1186/s12931-019-1096-9) contains supplementary material, which is available to authorized users.

Nasal intermittent positive pressure ventilation (NIPPV) holds great potential as a primary ventilation support method for Respiratory Distress Syndrome (RDS). The use of NIPPV may also be of great value combined with minimally invasive surfactant delivery. Our aim was to implement an in vivo model of RDS, which can be managed with different non-invasive ventilation (NIV) strategies, including non-synchronized NIPPV, synchronized NIPPV (SNIPPV), and nasal continuous positive airway pressure (NCPAP). Forty-two surfactant-depleted adult rabbits were allocated in six different groups: three groups of animals were treated with only NIV for three hours (NIPPV, SNIPPV, and NCPAP groups), while three other groups were treated with surfactant (SF) followed by NIV (NIPPV+SF, SNIPPV+SF, and NCPAP+SF groups). Arterial gas exchange, ventilation indices, and dynamic compliance were assessed. Post-mortem the lungs were sampled for histological evaluation. Surfactant depletion was successfully achieved by repeated broncho-alveolar lavages (BALs). After BALs, all animals developed a moderate respiratory distress, which could not be reverted by merely applying NIV. Conversely, surfactant administration followed by NIV induced a rapid improvement of arterial oxygenation in all surfactant-treated groups. Breath synchronization was associated with a significantly better response in terms of gas exchange and dynamic compliance compared to non-synchronized NIPPV, showing also the lowest injury scores after histological assessment. The proposed in vivo model of surfactant deficiency was successfully managed with NCPAP, NIPPV, or SNIPPV; this model resembles a moderate respiratory distress and it is suitable for the preclinical testing of less invasive surfactant administration techniques.