## 4 Discussion
Additionally, a surfactant fraction ranging between 25 and 30% was not recovered from the in vitro set-up elements and was most probably released as fugitive aerosols to the ambient air due to the air leaks between the NCs and the nares of the PrINT model. Consequently, the mean lung doses achieved with the Westmed and Fisher & Paykel NCs were dramatically reduced to 0.5% and 1.8% of the nominal dose, respectively. In previous benchmark studies, testing the same drug/device combination in a neonatal nCPAP circuit, we achieved lung doses above 10% [28; 37], which correlated well with the lung deposition obtained in scintigraphy studies conducted in neonatal piglets managed with nCPAP (mean surfactant lung deposition of 15.9 \(\pm\) 11.9%) [17]. These differences between nCPAP and HFNC may be partly explained by the position of the nebulizer within the setup, which was placed as close to the patient as possible during nCPAP experiments, between the nasal prongs and the connection of the ventilator circuit, to avoid aerosol dilution by bias flow. Conversely, in the present study, the nebulizer was placed right before the NCs, which promoted the impaction of aerosol particles within the narrow NC tubing, thereby reducing the lung dose. Moreover, it is worth mentioning that the bias flow was lower during CPAP experiments (5 L/min vs. 8 L/min in the present study) and that air leaks at the patient interface were minimized during nCPAP experiments by sealing the CPAP interface to the nares of the PRiNT cast with silicon, whereas in the present study, air leaks around the nasal interface were allowed. The delivery of a nominal dose of surfactant of 400 mg/kg to surfactant-depleted rabbits during HFNC did not show any beneficial effects in terms of arterial oxygenation or lung mechanics, in line with untreated controls. The 400 mg/kg dose was chosen based on the observation by [PERSON] et al., who described a pulmonary improvement in preterm lambs with as low as 2 mg/kg of surfactant depositing in the lungs after nebulization [38], an intrapulmonary surfactant dose strikingly lower compared with the currently approved surfactant instillation protocols (100-200 mg/kg). The different distribution patterns may explain a higher efficacy of lower intrapulmonary surfactant doses after nebulization; instilled surfactant tends to follow gravity, whereas surfactant aerosols follow ventilation. Therefore, with an expected lung deposition of 0.5-1.8%, one could estimate an intrapulmonary surfactant dose ranging between 2 and 8 mg/kg with a 400 mg/kg nominal dose, in the range of the intrapulmonary surfactant dose reported by [PERSON] et al. to elicit a lung response. However, as previously reported by [PERSON] et al. [35], the lung dose determined in vitro may have overestimated the actual surfactant deposition in spontaneously breathing rabbits. The outcomes of the present study contrast with previous studies in surfactant-depleted adult rabbits and neonatal piglets managed with nCPAP in which nominal doses of nebulized surfactant as low as 100 mg/kg achieved a slight but significant improvement of PaO\({}_{2}\)[13], and doses of 200 and 400 mg/kg were associated with a pulmonary response equivalent to the intratracheal instillation of a clinical dose of surfactant (200 mg/kg) in terms of gas exchange and lung mechanics [13; 15]. Thus, the in vivo results obtained in the present study indicate that nCPAP outperforms HFNC as an NRS type for surfactant administration with the current drug/device combination. This study has some limitations. We used commercially available NCs designed for neonates, which were applied to adult rabbits with different upper airway anatomy. Therefore, a flow of 8 L/min was required to achieve a pharyngeal pressure equivalent to that recorded during nCPAP at 5 cmH\({}_{2}\)O. Lower flows could likely achieve slightly higher lung doses.