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We investigated whether potentially injurious transpulmonary pressure could be generated by strong spontaneous breathing and exacerbate lung injury even when plateau pressure is limited to <30 cm H2O.Prospective, randomized, animal study.University animal research laboratory.Thirty-two New Zealand White rabbits.Lavage-injured rabbits were randomly allocated to four groups to receive low or moderate tidal volume ventilation, each combined with weak or strong spontaneous breathing effort. Inspiratory pressure for low tidal volume ventilation was set at 10 cm H2O and tidal volume at 6 mL/kg. For moderate tidal volume ventilation, the values were 20 cm H2O and 7–9 mL/kg. The groups were: low tidal volume ventilation + spontaneous breathingweak, low tidal volume ventilation + spontaneous breathingstrong, moderate tidal volume ventilation + spontaneous breathingweak, and moderate tidal volume ventilation + spontaneous breathingstrong. Each group had the same settings for positive end-expiratory pressure of 8 cm H2O.Respiratory variables were measured every 60 mins. Distribution of lung aeration and alveolar collapse were histologically evaluated. Low tidal volume ventilation + spontaneous breathingstrong showed the most favorable oxygenation and compliance of respiratory system, and the best lung aeration. By contrast, in moderate tidal volume ventilation + spontaneous breathingstrong, the greatest atelectasis with numerous neutrophils was observed. While we applied settings to maintain plateau pressure at <30 cm H2O in all groups, in moderate tidal volume ventilation + spontaneous breathingstrong, transpulmonary pressure rose >33 cm H2O. Both minute ventilation and respiratory rate were higher in the strong spontaneous breathing groups.Even when plateau pressure is limited to <30 cm H2O, combined with increased respiratory rate and tidal volume, high transpulmonary pressure generated by strong spontaneous breathing effort can worsen lung injury. When spontaneous breathing is preserved during mechanical ventilation, transpulmonary pressure and tidal volume should be strictly controlled to prevent further lung injury.