In the present study we found that mechanical ventilation with lower tidal volumes than 10 ml/kg and the loss of PEEP were associated with worse lung function and increased expression of inflammatory mediators in a murine model.
A large number of studies have used high tidal volumes (20-30 ml/kg) to investigate the effect of excessive alveolar distension and ventilator induced lung injury in mouse models. Those studies demonstrated that high tidal volumes can induce inflammation, barrier disruption, and pulmonary edema [1, 4–9, 12–15]. On the other hand low tidal volumes used in protective ventilation may lead to atelectasis and hypoxia . Data on the effect of very small tidal volumes in mechanical ventilation in murine models are sparse. In our study we sought out to investigate the effect of tidal volumes as low as recommended by the ARDS network study as being protective (6 ml/kg; ). This is below the tidal volume of 10 ml/kg that is commonly used as reference or protective ventilation in mouse models to compare the effects of low and high tidal ventilation [12–15] but it is in the range of normal tidal volumes of spontaneously breathing animals estimated on the basis of data from plethysmographic studies [16, 17]. Our aim was to specifically examine changes in hemodynamic parameters, lung function measurements and inflammatory cytokine expression in order to evaluate whether lower tidal volumes would be less injurious. Moreover, we sought to expand the knowledge on ventilator-induced changes in mouse lungs with low tidal volumes.
Lungs of mice and humans are different in terms of size, structure, and functional behaviour. The normal frequency of breathing is much higher mice (up to 200 breaths/min) compared to humans. Minute ventilation mainly depends on frequency since tidal volumes are small (5 to 15 ml/kg; [16, 17]). Therefore it was possible that ventilation with very small tidal volumes resulted in inadequate minute ventilation volumes causing hypoxia. On the other hand data from the ARDSnet demonstrated that ventilation close to physiologic parameters is the least injurious . However, we did not perform experiments with different respiratory rates which is a limitation of the study.
In the present study low tidal volumes were associated with very low oxygen saturation during mechanical ventilation. The most possible reason for this is that small tidal volumes (5 and 7 ml/kg) did not recruit sufficient alveolar units for adequate oxygenation. However, the tidal volumes we used are in the range of tidal volumes of spontaneously breathing mice [16, 17]. Moreover, there was no significant difference compared to a Vt of 10 ml/kg. Low tidal volumes may have caused atelectasis. Cycling opening and re-opening of the lung during the formation of atelectasis may cause increased shear forces thus leading to inflammation. Part of this effect could probably be prevented by application of PEEP. With the addition of PEEP oxygen saturation improved. Although we did not measure the lower inflection point it seems that the PEEP used in our study was high enough based on previous data from the literature . However recent studies showed that higher PEEP values are not injurious to healthy mice [19, 20].
With very small tidal volumes it is possible that gas exchange is hampered because of death space ventilation. Small volumes may not lead to adequate exchange of air in the alveoli if they are close to the death space. Although we cannot rule out the possibility that small tidal volumes led to hypoxia mostly due to death space ventilation we do not think that this was the main reason. First the mice used in our experiments were tracheotomized which minimizes anatomical death space. Second the system we used (Flexivent®) is constructed for the ventilation of small animals with small volumes. Death space in the system is very small.
In the present study the pulse rate did not significantly differ between the groups at baseline and after 30 min of ventilation. Although this is not a proof these data suggest that hemodynamics were not significantly altered by different tidal volumes. However, ventilation could be performed longer in the groups with a Vt of 10 ml/kg with or without PEEP compared to all other groups until criteria to stop ventilation were met (data not shown).
Resistance (Rn) increased during ventilation if no PEEP was applied but this effect was not statistically significant. In contrast Rn remained stable with the application of PEEP. However, Rn represents mostly the central airways. A significant increase in tissue damping (G) reflecting tissue resistance after 30 min of ventilation was observed. Our data agree with a study by Wilson and co-workers . In that study resistance showed a significant increase with high tidal volume ventilation and a small not significant decrease with low tidal volume ventilation (9 ml/kg) .
We observed a significant increase in tissue elastance (H) after 30 min of ventilation. This has also been described in a study by Allen and co-workers . In that study deep inflation has been shown to be beneficial when applied several times a minute. In our study we did not use deep inflation to recruit parts of the lung because we wanted to examine specifically the effect of small tidal volumes on lung function and cytokine expression without additional shear forces. Interestingly, tissue elastance was significantly lower after 30 min in the group with a Vt of 10 ml/kg and PEEP compared to all other groups.
Histological measurements revealed increased septal thickening in ventilated mice (data not shown). This finding most probably reflects formation of edema and influx of cells due to mechanical ventilation as been described in previous studies [22, 23]. We observed a marked increase in the numbers of PMN in ventilated lungs compared to spontaneously breathing animals. Influx of PMN due mechanical ventilation has been described previously [24, 25]. The application of PEEP had no significant effect on the numbers of PMN.
To further analyze the effect of low tidal volume ventilation on inflammation the expression of the neutrophil attractant cytokine MIP-2 and the proinflammatory cytokine TNFα was investigated. The increase in proinflammatory cytokine expression in groups with lower tidal volumes agree with previous data from the literature. Caruso and co-workers showed that ventilation with low tidal volumes caused similar proinflammatory and profibrogenic responses as high tidal volume ventilation compared to spontaneously breathing rats . However, in that study the authors did not apply PEEP. In a recent study by Terragani and colleagues  low tidal volume ventilation was not associated with increased inflammation. However, in that study human patients were investigated and extracorporeal carbon dioxide removal was used. Agreeing with our data Nakos and co-workers demonstrated in increased numbers of PMN in the lungs of mechanically ventilated patients with atelectasis .
We observed a marked reduction of proinflammatory cytokine expression with the application of PEEP. Small tidal volumes may cause inflammation due to formation of atelectasis and increased shear forces during opening and re-opening of alveoli. It is not surprising that the application of PEEP that prevents the formation of atelectasis can reduce shear forces and thereby decrease expression of inflammatory mediators.
In our experiments mice were ventilated for 30 min. This has to be considered as a very short time frame. However, changes in lung function parameters, in neutrophil influx, and in cytokine expression could be observed compared to spontaneously breathing animals. Moreover, this time frame enabled us to apply different tidal volumes without additional oxygen to stabilize oxygen saturation. This minimizes a potential confounding effect of oxygen.
Oxygen saturation was very low in the animals with very small tidal volumes. Some of them were hypoxic or were exposed to severe hypoxia (oxygen saturation below 75% at baseline). Unfortunately, we did not perform blood gas analysis. However, inflammation was mostly increased with very low tidal volumes. Therefore hypoxia may not be the only cause for changes in inflammatory markers. We cannot clearly comment on survival issues because ventilation was terminated if there was a significant drop in oxygen saturation or reduced pulse rate prior to death of the animal. Based on the ventilation time in the different groups it has to be concluded that survival was probably shorter in the groups that were ventilated with either a Vt of wither 5 ml/kg or 7 ml/kg compared to the group with a Vt of 10 ml/kg regardless whether PEEP was applied or not.
We also looked at a group that was ventilated with a tidal volume of 30 ml/kg as a control for high-tidal ventilation. This group had elevated PMN numbers and significantly increased proinflammatory cytokine expression as expected from data from the literature (data not shown). Our data support the notion that both too low and too high volumes in mechanical ventilation lead to lung damage.