Overview over principle findings
This is to our knowledge the first study examining the impact of weaning methods PSV and SBT on diaphragm function and metabolism after the onset of VIDD. Our data show that both strategies further reduced diaphragm force despite the fact that they reversed oxidative stress and proteolysis caused by mechanical ventilation.
These findings are discussed in detail below.
Weaning using SBT and PSV does not result in diaphragm function recovery
In the current study, neither SBT nor PSV applied as weaning strategies after 12 h of CMV were associated with diaphragm function recovery. In fact, each strategy led to a worsening of diaphragm function and intriguingly the magnitude of this attenuation was similar whether the diaphragm was gradually (PSV) or intermittently fully (SBT) reloaded. Our data are surprising in the context of previous studies, which demonstrated a rapid regaining in diaphragm force within 4–7 to 24 h after induction of VIDD, when the animals breathed completely independent from further assistance but with the risk of muscle overload and inflammatory damage [
9,
11]. Importantly, in the PSV group the inspiratory pressure delivered by the ventilator was decreased, i.e. the diaphragm had to contract more forcefully; obviously, the increase of work was not sufficient to regain force.
Although 5 min of spontaneous breathing seem to protect, at least partially from a contractile deficit [
8], it is obviously not enough to regain muscle force. Martin and colleagues proposed in patients an inspiratory muscle training of very low duty cycles (i.e. 40 breaths per day), resulting in a better patient inspiratory force [
20]. From our data we cannot support these findings that a low duty cycle might be enough to regain muscle force.
Theoretically, this decline in force could be induced by overload; nevertheless the triggering was carefully set in the PSV mode and airway obstruction during the 5 min SBT phases were avoided.
SBT and PSV reverse diaphragmatic oxidative stress
MV resulted in an increase in oxidized proteins and lipid peroxidation making sarcomeres more susceptible for the breakdown by the protease system [
6]. This finding is in line with former publications from our groups [
6,
11,
21], defining it as one major pathway of VIDD [
22]. The reduction in oxidative damage during the weaning process might result in less cleavage of sarcomeric proteins.
Both weaning strategies were able to reverse the amount of oxidized proteins and lipid peroxidation but this did not result in diaphragmatic functional improvement. Muscle force depends on the number of myosin heads forming parallel cross bridges per half sarcomere, the steady state fraction of strongly bound cross bridges in the force generating state and the force generated by cross bridges [
23]. Mitochondrial uncoupling may be reduced by activation of any kind as in our model and result in decreased oxidative damage of cellular structures [
23], but this had no impact on force generation.
Weaning reverses the activation of the calpain system
Mechanical ventilation induced an elevation of calpain activity compared to CON in line with former publications from our group and others [
24,
25]. The increase in calpain activity was significantly down regulated after weaning by PSV compared to CMV, but not in SBT group. Previous studies examining diaphragmatic recovery after MV reported various effects on these proteases after reloading [
5,
26,
27]. Thus, full diaphragm reloading after extubation resulted in a complete reversal towards control levels of both proteases calpain and caspase-3 after 12 h of reloading and this was associated with normalization of diaphragm function [
11]. By contrast, Thomas and colleagues did not observe any amelioration of protease activity after 4–7 h of diaphragmatic reloading [
9], although animals in their study did most likely have less diaphragm reloading due to sedation and diaphragm fully recovered normal function. This suggests that the calpain system restoration is not only time dependent but also requires a certain amount of reloading to normalize. In addition, these observations also underline that the down regulation of these proteases is not required for diaphragm force to recover.
We could not detect changes in macrophagic or neutrophilic invasion between the groups in contrast to Thomas and colleagues. Possibly, different degrees of reloading cause subsequently different inflammatory responses.
Weaning did not alter the decrease in protein synthesis
As one major upstream between gaining of proteins (protein synthesis) and proteolysis acts AKT. It increases protein synthesis in the phosphorylated state and, while dephosphorylated, AKT can induce the autophagy pathway via mammalian target of Rapamycin [
28,
29]. In the current study, a decrease of pAKT/AKT was observed during mechanical ventilation, comparable to former studies [
28]. This ratio remained reduced after both weaning periods, indicating that protein synthesis was still impaired with diaphragm reloading regardless of the load applied to the diaphragm. It is therefore unlikely that it contributed to the worsening of diaphragm function seen after weaning.
Model validation
Gradual reloading is used to protect the muscle from overload-induced damage and to increase diaphragmatic reloading while reducing inspiratory pressure support [
30]. We designed the PSV group closely to clinical needs while beginning with support-level identical to the inspiratory pressure during MV, gradual reduction, and adaption based on respiratory physiology [
13]. By decreasing pressure support, we surely induced an increase in diaphragmatic activation. We re-increased the pressure level during PSV when the respiratory rate increased above 100 and/or hypercapnia occurred. It was impossible in this model due to the ventilator type to measure exact tidal volumes to calculate Vt/RR quotient or assess distress as this can be done in awake patients breathing through a tracheal cannula. Therefore our approach to guide the PSV setting was close to but not exactly mimicking the clinical situation during the weaning process. Nevertheless, even if the ventilator is set in the best clinical practice in patients undergoing pressure support ventilation, over-assistance in the clinical setting may be apparent [
31]. Over-assistance may be most likely the reason for the worsening of diaphragm function in our study.
The frequency and duration of the spontaneous breathing trials was chosen closely to the work of Gayan-Ramirez and others, demonstrating preservation from VIDD even by these short terms of interruption from mechanical ventilation [
8]. However, even in the beginning of a weaning process, short trials of max 5 min several times a day may be used to initiate weaning from the ventilator. It is important to notice that our approach to implement hourly spontaneous breathing intervals was based on scientific reasons by Gayan-Ramirez and colleagues demonstrating preservation of myogenic signalling using this strategy (see above) [
8] and cannot mimic the clinical scenario of a usually 30 min lasting first spontaneous breathing trial that is prolonged in the following weaning attempts [
32].
Limitations of the model and clinical impact
We chose our established rat model to investigate the effects of different reloading approaches after MV, those are appropriate for the animal model but differ from those used in clinical practice. There is, however, evidence that the same pathways are activated in rats and humans and therefore findings in these animal models can be transferred to humans [
33,
34]. However, our model is the first to describe biological effects of gradual vs. intermittent reloading on the diaphragm, which is not proven in humans, yet.
Over-assistance during PSV and due to the spontaneous breathing trial duration might have influenced these results. Hudson and colleagues revealed that over-assistance in pressure support ventilation cannot prevent from VIDD [
35] and could not reproduce the findings of Futier and colleagues that demonstrated preservation of diaphragm metabolism by pressure support ventilation [
36]. The actual load might therefore be essential for the effects on diaphragm function. Nevertheless, prevention, i.e. remaining sufficient load is different than regaining of force as in our model.
Additionally we have to address the influence of the sedative on the respiratory effort. Vaschetto and colleagues impressively demonstrated that the breathing effort during pressure support ventilation is dose-dependent of the sedative agent [
37]. Our animals were sedated keeping them in a state of low sedation that suppressed moving and vegetative disturbance. However we could not monitor or titrate this sedation compared to the mentioned study [
37].
We decided not to add another control group of 24 h of mechanical ventilation. Studying the rats under mechanical ventilation after 24 h of mechanical ventilation to address, whether weaning would improve the loss of diaphragm force caused by 12 h mechanical ventilation, may not be fully suitable. Indeed, after 12 h of mechanical ventilation, diaphragm force is reduced and we wanted to know which weaning approach would lead to more efficient restoration of diaphragm function, when weaning the rats at this time point.
Additionally, the weaning period of 12 h was chosen after the discrepant results from our study [
11] and Thomas et al. [
9] study, which have been described above. In our study investigating recovery with full diaphragm load [
11], we have explained that the on-going decline in contractile force after 12 h of full load might have been due to overload that would be absent in a gradual reloading as the one chosen in the actual work. The decline in force despite loading is from our perspective due to insufficient loading.
Additionally protease activation, removal of oxidized proteins and restoration of function may also be influenced by time; reduction of protease action may be a first step to re-balance cellular homeostasis, but contractile force needs intact –replaced- sarcomeres to act. Therefore our results can only be interpreted in the investigated time frame of unloading and reloading.
Does this study help to understand what happens in weaning patients? At first, patients are weaned in more than 12 h and even severely ill individuals can get weaned. Importantly, our data indicate that the amount of load is decisive for weaning success.