Approximately 12% of births are accompanied by foetal passage of meconium in utero (1, 2). Of these, 4% are complicated by the development of meconium aspiration syndrome (MAS) in the newborn, a potentially devastating respiratory condition with high associated morbidity and mortality (3). The initiation of foetal inspiratory efforts or ventilation with positive pressure during resuscitation, together with the onset of spontaneous breathing in the newborn, causes the meconium present in the airway to produce partial or complete airway obstruction leading to the coexistence of areas of atelectasis and emphysema. Atelectasis causes ventilation-perfusion (V/Q) mismatch, whilst alveolar overdistention explains the high incidence of air-leak syndrome in this condition. In vitro studies on calf lung surfactant have shown that meconium also inactivates surfactant in a dose-dependent manner (4). Furthermore, meconium is a highly irritative substance that induces lung inflammation, which plays a key role in the pathophysiology of MAS-induced lung damage, increasing parenchymal oedema, impairing endothelial function, and spreading the damage to lung areas not exposed to meconium (2, 5, 6). Post-resuscitation reoxygenation contributes to tissue damage, inflammation and oxidative stress with damaging consequences (1, 2). One of them, of crucial importance for clinical management and survival, is pulmonary hypertension (PH), which causes severe hypoxemia and may negatively affect myocardial function and cardiac output (1). The combination of all these mechanisms translates clinically into hypoventilation, hypoxemia and tissue hypoperfusion, with the need for aggressive mechanical ventilation, high concentrations of oxygen, inhaled nitric oxide (iNO), haemodynamic support and extracorporeal membrane oxygenation (ECMO) in the most severe cases (3).

There is no specific treatment of MAS other than lung lavage with surfactant, but the efficacy and safety of this procedure is controversial (1). Surfactant without lavage and iNO offer some beneficial effects in selected cases (7). Thus, management is mostly supportive, dealing with ventilatory and hemodynamic problems, and preventing complications such as brain damage (7). Steroids did show some promise in preclinical studies as a therapeutic tool against MAS-induced inflammation, but this failed to translate into clear benefits in randomised controlled trials (RCT) (5). In addition, steroids increase the risk of infectious diseases, which can be a concerning complication (5). This, together with the concern for the deleterious effects on neurodevelopment associated with the use of steroids in the neonatal period, make such therapy problematic (7).

Cannabidiol (CBD) is a non-euphoric phytocannabinoid, and its administration across a range of animal models of disease is known to be associated with anti-inflammatory properties (8) including decreased lung inflammation in a murine model of LPS-induced lung damage (9) and reduced oxidative stress (10). Further, studies from our group have demonstrated that CBD 1 mg/kg i.v. was associated with reduced HI brain-induced inflammation-mediated lung damage in piglets (11). The mechanisms of action of CBD are not fully elucidated, but preclinical studies suggest that CBD possesses a diverse polypharmacology including antagonism of G protein-coupled receptor 55, desensitization of transient receptor potential cation channel subfamily V member 1 and activation of 5-HT_1A receptors (12, 13). Despite the lipophilic nature of CBD, our group has demonstrated that CBD can be administered by i.v. route using an emulsion of CBD on saline, ethanol and Solutol^R (11, 14–16). Administration of CBD 1 mg/kg by i.v. bolus to newborn piglets leads to serum C_max = 263 ± 41 ng/mL with T_max = 0.15 h and t1/2 = 1.09 ± 0.27 h (14, 16).

This study aims to test the effects of CBD in a model of another inflammation-based lung disease, MAS.

The experimental protocol met European and Spanish regulations for protection of experimental animals (86/609/EEC and RD 53/2013) and was approved by the Ethics Committee for Animal Welfare from the University Hospital Puerta de Hierro Majadahonda (Madrid, Spain) and Hospital Clínico San Carlos (Madrid, Spain).

In this study we demonstrate that post-insult administration of CBD exerts beneficial effects in a piglet model of MAS. A striking result was that CBD treatment after MAS exerted a beneficial effect on ventilation, with less elevated pCO2 levels and normal pH, but CBD did not improve oxygenation, with similar FiO2 requirements and OI in MAS + VEH and MAS + CBD piglets throughout the experimental period. Oxygenation problems in MAS are a consequence of impaired diffusion due to V/Q mismatch caused by different factors, including the presence of non-ventilated atelectatic areas as a result of mechanical obstruction of the airways by meconium and alveolar occupation by edema, inflammation or meconium, as well as thickening of the blood gas barrier caused by inflammation (1, 2). In MAS, V/Q mismatch is not homogeneous in lung parenchyma since meconium is not evenly distributed, leading to areas with more inflammation than others (1, 2). V/Q inequality disrupts the transfer of all gases in lung including O₂ and CO₂ (18). We speculate that the anti-inflammatory effect of CBD decreased the V/Q inequality derived from an uneven distribution of inflammation in the lung after MAS and thinning of the blood-gas barrier, resulting in improved gas diffusion. Such hypotheses are supported by the fact that CBD’s effects on pCO₂ were achieved despite lower MV and Vt values in MAS + CBD piglets as compared to MAS + VEH animals. The different dissociation curves of O₂ and CO₂, however, determines that improvements of V/Q inequality results in improvements in ventilation earlier than in oxygenation (18). Increasing pulmonary blood flow in the presence of ventilation-perfusion inequality also improved ventilation before improving oxygenation (18). CBD improves pulmonary blood flow at doses that do not affect systemic hemodynamics (19) but this was not assessed in our experiments. Therefore, it is possible that a longer follow-up would be required to detect an improvement in oxygenation in our model. Hypoxemia after MAS can also be a consequence of secondary pulmonary hypertension (1, 2), but this condition was not assessed in our study.

Meconium aspiration syndrome-induced lung damage was demonstrated in the histological analyses, with increased leucocyte infiltration, parenchymal oedema and haemorrhage in MAS-VEH lungs, features that have been attributed to a meconium-induced inflammatory reaction in lungs (17). In agreement, we observed that histological injury was associated with increased production of TNFα, a well-documented finding in piglet models of MAS (5). CBD treatment reduced MAS-induced lung damage, reducing leucocyte infiltration, parenchymal oedema and haemorrhage. These beneficial effects were consistent with the prevention of MAS-induced increases in TNFα concentration by CBD. These results agree with previous studies demonstrating anti-inflammatory properties of CBD on the lung, in murine models of LPS-induced lung damage as well as in cerebral hypoxia-ischemia-mediated distant lung injury in newborn piglets (9, 11). The present results, however, are a more robust demonstration of the anti-inflammatory effect associated with CBD in the lung considering MAS is a lung disease with a particularly strong inflammatory component (2, 5, 6). Thus, although CBD shows some effects that could theoretically account for lung protection after MAS, such as the improvement of pulmonary blood flow at doses that do not affect systemic hemodynamics (19) and the modulation of oxidative stress (10), the paramount importance of inflammation in MAS pathophysiology points to the anti-inflammatory effects as the main component responsible for the protective effect of CBD in MAS. In fact, known anti-inflammatory substances such as steroids, in particular prednisolone and dexamethasone, administered shortly after meconium infusion in newborn pigs and rabbits, lead to similar beneficial effects, improving gas exchange, decreasing ventilatory pressures and reducing lung oedema (5). Therefore, CBD achieved similar anti-inflammatory-based beneficial effects to steroids without significant side effects.

In fact, CBD treatment prevents the MAS-induced progressive fall in MABP, which was significant in vehicle-treated piglets, although no supportive inotropic treatment was initiated since MABP did not fall below 30 mmHg. If the duration of this study was extended inotropic support may well have been required. This effect was similar to that reported for piglets after a hypoxic-ischaemic insult (14, 15). Such beneficial effects of CBD on blood pressure in two different neonatal models are reassuring and open an interesting possibility for CBD as a part of the treatment of hemodynamic instability in critically ill neonates. Interestingly, MAS + CBD piglets showed increased HR starting shortly after CBD administration, which remained stable throughout the experimental period. Such an effect was not observed in HI piglets receiving CBD 1 mg/kg i.v. (14, 15). It remains to be elucidated if this effect is a consequence of the higher dose of CBD used in the present study or if this difference resulted from using CBD in a different condition. The clinical relevance of this effect is likely to be limited, since it did not translate into hemodynamic instability with CO and MABP remaining unaffected.

Our study presents several shortcomings. The main limitation of our study is that MAS was induced at a postnatal age of 2–4 days, so the animals had probably completed cardiopulmonary transition by that time. In a more translational model MAS results from prenatal meconium aspiration during gasping after inducing asphyxia, which is likely to leads to lung injury and lung vessel reactivity changes more similar to the actual condition in human newborns (20). However, in our model piglets showed greater ventilatory and hemodynamic instability than lambs over a similar follow-up period (20), which might represent an advantage when the aim is to test the efficacy of a novel therapy. In this regard, although the presence of pulmonary hypertension was not assessed and consequently the effect of CBD on it could not be evaluated, its contribution to hypoxemia in our model was less likely. On the other hand, the role of surfactant inactivation in our model was not studied and thus the effect of CBD on this pathophysiological mechanism could not be assessed either. Finally, studies with longer time of follow-up would be necessary to get more insights into the mechanisms of CBD beneficial effects as well as the true translational value of the treatment.

The dose of CBD used in this study was based on the fact that 1 mg/kg CBD has demonstrated efficacy in mild- to moderate- models of HI in newborn rats, mice and piglets, but not in severe models of HI in newborn piglets (21). The model of MAS employed in this study produces a profound inflammatory insult so it was hypothesized that a higher dose of CBD might be required; furthermore, 5 mg/kg CBD has shown beneficial effects in rodent models of newborn and adult ischemic stroke (10). However, future studies could explore the potential efficacy of lower and higher doses of CBD in this model of MAS.

In conclusion, CBD administered after MAS induction in newborn piglets improved ventilation and respiratory acidosis with lower ventilatory requirements. These effects were associated with the reduction of histological lung damage in a manner related to modulation of inflammation. CBD treated animals were hemodynamically more stable without significant side-effects. This constitutes an asset for CBD when compared to other anti-inflammatory therapies (e.g., steroids). More studies are needed to further characterize the effects of CBD on MAS.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Ethics Committee for Animal Welfare from the University Hospital Puerta de Hierro Majadahonda (Madrid, Spain) and Hospital Clínico San Carlos (Madrid, Spain).

LA and JM-O contributed to the conception and design of the study and wrote the first draft of the manuscript. LA, LB, EV, MR, and JM-O performed the experiments. LA, LB, AD, and JM-O performed the biochemical and histological studies. JM-O and WH performed the statistical analysis and organized the database. WH edited the final version of the manuscript. All authors contributed to manuscript revision and read and approved the submitted version.

JM-O had a Research Agreement with GW Research Ltd. (Cambridge, United Kingdom) when these experiments were performed. WH was an employee of GW Research Ltd. (Cambridge, United Kingdom) where he received salary and held shares. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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