Early Restrictive Fluid Strategy Impairs the Diaphragm Force in Lambs with Acute Respiratory Distress Syndrome

This study was part of an extensive experiment with the same research design but with different research questions all focused on the cardiopulmonary effects of fluid strategy during experimental pediatric ARDS.¹⁷  The current study focused on the effects of a restrictive fluid strategy versus a liberal fluid strategy on diaphragm structure and function. The effect of a contraction stimulus on the transdiaphragmatic pressures and the formation of diaphragmatic muscle fiber edema were the primary outcomes. Secondary outcomes were changes in diaphragmatic microcirculation, inflammation, and oxidative stress and the direct effect of the application of PEEP on the transdiaphragmatic pressures.

Procedures involving animal data, anesthetics, mechanical ventilation, and surgical techniques were performed in accordance with previously described methods.¹⁷  See appendix in the Supplemental Digital Content 1(https://links.lww.com/ALN/C802) for further details. Experimental details concerning the current study are mentioned separately in this article.

This study was approved by the local ethics committee on animal research of the Radboud University Medical Center (Nijmegen, The Netherlands; license No. RU-DEC 2016-0089) and was performed in accordance with the Dutch and European legal requirements on the use and protection of laboratory animals. The experimental procedures were designed to minimize the number of animals used, as well as to minimize animal suffering. The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for animal research were followed.¹⁸ 

Twenty-one female lambs with a mean weight of 12.7 kg (95% CI, 10.6 to 14.8) and approximately 2 to 6 weeks of age were studied. The lambs were of the Texelaar–Flevolander breed, except one that was a Romanov lamb due to unavailability of a Texelaar–Flevolander lamb. Only female lambs were used as precise urine production monitoring (in the context of fluid balance) was important for this study, and urethral catheterization is more difficult in male lambs.

For the estimation of pleural pressure,¹⁹  an air-filled balloon catheter (5-French, Cooper Surgical, USA) was positioned in the esophagus. Catheter positioning and validation were performed according to a recent consensus statement.²⁰  Briefly, after positioning, the balloon was inflated with 1 ml of air.^21–23  The intragastric position of the balloon was confirmed by the visualization of positive deflections of balloon pressure during external compression of the left upper abdominal quadrant. Subsequently, the catheter was withdrawn until cardiac artefacts appeared on the pressure tracings, indicating that the balloon was placed in the lower third of the esophagus.

Equilibration of the system with ambient pressure was performed before each measurement (at baseline and after 6 h). To validate the correct position of the balloon, a dynamic occlusion test was performed. This was performed by measuring the ratio of change in esophageal pressure (ΔP_eso) to the change in airway opening pressure (ΔP_aw) during three spontaneous inspiratory efforts against a closed airway at end expiration. Catheter inflation was acceptable if ΔP_es/ΔP_aw ratio was between 0.8 and 1.2.^19,24 

Via a small laparotomy of 1 to 2 cm, a second pressure balloon catheter (5-French, Cooper Surgical, USA) was placed in the right upper abdominal cavity for measuring intraabdominal pressure, after which the abdominal wall was closed. Transdiaphragmatic pressures were used to estimate the force-generating capacity of the diaphragm and were calculated as transdiaphragmatic pressure (cm H₂O) = intraabdominal pressure (cm H₂O) – P_eso (cm H₂O).

The phrenic nerves were stimulated at baseline and after 6 h of mechanical ventilation by bilateral transvenous stimulation with a fixed frequency of 40 Hz, a fixed pulse width of 210 μs, an intermittent stimulation of 1 to 2 s on and 2 to 3 s off,²⁵  and a fixed supramaximal stimulation (70 V). For this reason, an electric stimulation catheter (IBI-81102, decapolar 6 French, electrode spacing: 2-5-2 mm, St. Jude Medical, USA) was placed in the brachiocephalic vein via the left internal jugular vein for bilateral transvenous phrenic nerve pacing. In feasibility experiments before the current study, the optimal position of the catheter was determined with the pacing electrodes at the transition of the brachiocephalic and superior caval vein as, in lambs, the left and right phrenic nerves are positioned close to each other at that point. At this point, maximal bilateral stimulation of the diaphragm was obtained, leading to maximal negative esophageal pressures and clinically visual bilateral diaphragm contraction. The stimulation catheter was connected via an 8-pin extension cable (model 990066, Medtronic Inc., The Netherlands) and electrode switch box (model 19038, Medtronic Inc.) to an external neurostimulator (model 37022, Medtronic Inc.) and programmed via an N’Vision clinician programmer (model 8840, Medtronic Inc.).

For quantification of the microcirculation of the diaphragm, a video microscope based on incident dark-field (IDF) imaging (Cytocam, Breadius Medical, The Netherlands) was placed at the left side of the diaphragm via the abdominal cavity by reopening the closed laparotomy at the end of the experiment before euthanasia. For optimal imaging of the diaphragm, transparent silicone oil (Oxane 5700, Bausch and Lomb, USA) was placed on the surface of the video microscope. Five sequences of steady 10-s clips were obtained from the diaphragm microcirculation while avoiding pressure artefacts with the video microscope.

A blinded investigator (L.M.v.L.) scored the captured IDF clips according to the Microcirculation Image Quality Score defined by Massey and Shapiro²⁶  and Massey et al.²⁷  In short, the images were scored on six categories: illumination, duration, focus, content, stability, and pressure. The videos were assigned a score of 0 = good, 1 = acceptable, or 10 = unacceptable for each category. Any video with a composed score of 10 or higher was discarded from future analysis.

Image acquisition was performed according to published consensus criteria.²⁸  The videos were assessed on microvascular flow index and microvascular density. Quantification of flow (microvascular flow index) was based on determination of the predominant type of flow in four quadrants, categorized as 0 = no flow, 1 = intermittent flow, 2 = sluggish flow, and 3 = continuous flow. The values of the four quadrants were averaged. Microvascular density was calculated as the number of vessels crossing arbitrary lines divided by the total length of these lines (i.e., number of crossings).

Ventilation parameters were acquired using Servo Tracker (version 4.1, Maquet, Sweden), a software tool for the collection and presentation of performance data from Servo-i (Maquet). These recorded signals were analyzed using custom-written MATLAB scripts (Matlab R2017b, MathWorks Inc., USA).

Plateau pressure (P_plat) was determined as the airway pressure when the flow became 0 during an inspiratory occlusion. Total PEEP (PEEP_tot) was determined as the airway pressure when flow became 0 during an expiratory occlusion. Driving pressure was defined as P_plat−PEEP_tot. Static lung mechanics were collected and defined as follows: lung compliance (ml/cm H₂O), tidal volume (V_T)/(P_plat−P_eso, end-inspiratory)−(PEEP_tot−P_eso, end-expiratory); chest compliance (ml/cm H₂O), V_T/(P_eso,end-inspiratory−P_eso, end-expiratory); airway resistance (cmH₂O/L/s), Peak airway pressure (P_MAX)−P_plat/flow; transpulmonary pressure at end exhalation (cm H₂O), PEEP−P_eso, end-expiratory. The hemodynamic parameters were collected using a PiCCO device (Pulsion Medical Systems, Germany) for measuring intraarterial blood pressure and cardiac output via a transpulmonary thermodilution method according to previously described methods.²⁹ 

The experimental protocol was previously described in detail.¹⁷  In summary, the animals (n = 19) were randomized into two groups receiving either a restrictive (n =10) or a liberal (n = 9) fluid regimen (fig. 1). Randomization was conducted by drawing from randomly generated treatment allocations within sealed opaque envelopes. Subsequently, these animals received intravenous oleic acid into the right jugular vein catheter to induce acute lung injury as a model for pediatric ARDS.^30,31  Also, two randomly chosen animals were taken as controls. These animals did not receive oleic acid to induce ARDS but only received anesthesia and mechanical ventilation to observe the effects of the anesthetics and mechanical ventilation on the studied outcome parameters.

During the 6-h study period of mechanical ventilation, the animals in the liberal group received fluids at a maintenance of 120 ml · kg^–1 · day^–1, and the animals in the restrictive group received 60 ml · kg^–1 · day^–1. In case of hemodynamic instability, defined as blood pressure and cardiac output at less than 80% of baseline values, the liberal group was resuscitated mainly with Ringer’s lactate and whole blood, and the restrictive group was resuscitated mainly with norepinephrine. During the experiment, the aim was to keep the hematocrit within the same range compared to baseline values in all animals. If the hematocrit declined 10% or more, a whole-blood transfusion was administered.

The animals were ventilated in a volume-controlled mode according to the principles of protective lung ventilation aiming V_T between 6 and 8 ml/kg and limiting inspiratory plateau pressures of less than 30 cm H₂O.³²  The Paco₂ target was set between 4.5 and 6.0 kPa by adjusting the ventilatory parameters with respect to a maximum V_T of 8 ml/kg. Permissive hypercapnia was accepted if the pH remained acceptable (greater than 7.20) to keep the V_T at or less than 8 ml/kg. PEEP was set according to the lower PEEP titration model according to the ARDS network ventilatory protocol.³³ 

It was expected that, in contrast to the restrictive group, the animals in the liberal fluid group would need higher levels of PEEP due to the development of pulmonary edema with subsequent compression atelectasis resulting in changes in the end-expiratory lung volume. Changes in the end-expiratory lung volume will influence the position of the diaphragm, subsequently affecting the force-generating capacity of the diaphragm.³⁴  Since we aimed to study the diaphragm function under equal circumstances, the transdiaphragmatic stimulation was performed at various levels of PEEP in both groups. In doing so, at baseline and after 6 h of mechanical ventilation, diaphragmatic stimulations were performed, during an occlusion test (expiratory hold) at PEEP levels of 5, 10, 15, and 20 cm H₂O. During these measurements, PEEP was applied in increasing order (PEEP 5-10-15-20 cm H₂O) and immediately afterward in reverse order (PEEP 20-15-10-5 cm H₂O) to evaluate consistency in the force-generating capacity of the diaphragm. The average of three measurements was calculated.

Before the end of the experiment (after 6 h of mechanical ventilation, just before euthanasia), diaphragmatic microcirculation was obtained. At the end of the experiment, the lambs were euthanized with intravenous pentobarbital (150 mg/kg). After euthanasia, the whole diaphragm was immediately dissected, and biopsies were obtained for histological measurements.

Full-thickness biopsies were obtained from the same region of the right anterior costal diaphragm lateral to the insertion of the phrenic nerve in all animals (Supplemental Digital Content 2, https://links.lww.com/ALN/C803). Muscle biopsies were frozen immediately in liquid isopentane for histochemistry and collected in 4% buffered formalin for paraffin embedding. Paraffin sections stained with hematoxylin and eosin were prepared from longitudinal sectioned muscle fibers. For histochemistry, transverse sections of the muscle fibers were used as these sections yield much more information than longitudinal sections for light microscopy.³⁵  Frozen cryostat sections (5 mm thick) were used for staining with hematoxylin and eosin and for immunohistochemistry using antibodies clones BAD-5 and Sc-71, directed against the slow (type 1) and fast (type 2) isoforms of myosin heavy chain, respectively. These cryosections were rehydrated for 10 min in phosphate buffer and subsequently blocked with phosphate buffer containing 0.3% (w/v) bovine serum albumin. Endogenous peroxidase activity was blocked by treatment in 3% hydrogen peroxide in phosphate-buffered saline for 20 min. After preincubation with phosphate buffer containing 0.3% (w/v) bovine serum albumin 1%, the cryosections were incubated with the primary antibody for 1 h at room temperature, rinsed in phosphate-buffered saline, and incubated with a biotin-conjugated rabbit antimouse antibody for 1 h at room temperature. Subsequently the sections were incubated with Avidin-Biotin Complex kits (VECTASTAIN PK6100, Brunschwig Chemistry, The Netherlands) for 1 h. The peroxidase activity was visualized by staining with diaminobenzidine as substrate.

To objectively quantify slow and fast twitch fibers, the Fiji image-processing package (Jug and Tomancak, Max Planck Institute of Molecular Cell Biology and Genetics, Germany) was used.³⁶  A Fiji macro was created and automatically applied to all images. The Fiji macro first uses median filtering and thresholding to separate the marked parts of the image from the background. This foreground mask is next processed by reducing the number of small holes that result from uneven marking in cells. Finally, a distance transform watershed is used to segment the foreground mask into individual cell segments, which are counted and measured. Manually annotated images were used to verify the macro output and to optimize the parameters used in the macro.

To determine inflammatory mediators and oxidative stress, 50- to 100-mg frozen cryostat diaphragm biopsies were dissolved in a tissue protein extraction buffer and protease inhibitor cocktail. After obtaining homogenate, the proinflammatory cytokines (interleukins 1β, 6, and 8 and tumor necrosis factor α) and anti-inflammatory cytokines (interleukin 10) were determined using a multiplex cytokine panel (Luminex kit; SCYT1-91K, MILLIPLEX ovine cytokine/chemokine panel 1, Merck Chemicals B.V., The Netherlands). The lower limits of quantification for the cytokines were 25.6 pg/ml (interleukin 1β), 15.4 pg/ml (interleukin 6), 1.54 pg/ml (interleukin 8), 96 pg/ml (tumor necrosis factor α), and 3.2 pg/ml (interleukin 10). Oxidative stress was determined by measuring malondialdehyde, which is produced due to the oxidation of fatty acids, using the thiobarbituric acid reactive substances assay kit (ZeptoMetrics, Bio-connect B.V., The Netherlands). The cytokine and malondialdehyde data were normalized to total protein content (measured using a bicinchoninic acid protein assay kit; Thermo Scientific, USA) and expressed as picogram per milligram protein (cytokines) or nanomoles per milligram protein (malondialdehyde).

Statistical analysis was performed with SPSS (version 25.0; IBM, USA) and GraphPad Prism (version 5.03; GraphPad, USA). Continuous variables were presented as medians (25th to 75th percentile) unless otherwise specified, and comparison between groups was performed by Mann–Whitney U tests.

Mixed model analysis was conducted for hypothesis testing in which we included a random intercept for sheep and accounted for repeated measurements and multiple inferences for PEEP. We adjusted for baseline values of PEEP and accounted for multiple testing (Bonferroni). The data are presented as the estimated marginal means with 95% CI.

The minimal sample size was taken to detect a difference in transdiaphragmatic pressure of 4 cm H₂O, with an SD of 2.6.³⁷  Considering an α error 0.05 and a power of 80%, a sample size of seven lambs was required in each randomization arm of the study. To compensate for possible dropouts, the sample size was increased with two to three lambs in each arm of the study. A two-sided P value of less than 0.05 was considered statistically significant. In the study, n refers to number of animals.

The data sets analyzed during the current study are available from the corresponding author on reasonable request.

Three of nineteen lambs died prematurely due to refractory circulatory deterioration after oleic acid induction. Because measurement of the transdiaphragmatic pressures at 6 h could not be performed in these animals, they were excluded from the final analysis. At baseline, mechanical ventilation settings were similar between the two groups, and both groups developed a clinical comparable moderate pediatric ARDS (table 1). As expected, the control animals (n = 2) did not develop clinical signs of ARDS, and the characteristics of these animals are portrayed in Supplemental Digital Content 3 (https://links.lww.com/ALN/C804). At the end of the experiment, the cumulative fluid balance in the liberal group (liberal vs. restrictive: 86.0 ml/kg vs. 23.3 ml/kg; P < 0.001) and the cumulative dose of norepinephrine in the restrictive group (restrictive vs. liberal group: 130.7 µg/kg vs. 64.9 µg/kg; P = 0.001) were significantly higher.¹⁷ 

The baseline transdiaphragmatic pressures for the individual lambs are shown in Supplemental Digital Content 4 (https://links.lww.com/ALN/C846). Overall, there was a significant difference for intervention (P < 0.05). The pressure-generating capacity of the diaphragm after 6 h of mechanical ventilation decreased more in the restrictive group (–9.6 cm H₂O [95% CI, –14.4 to –4.8]) compared to the liberal group (–0.8 cm H₂O [95% CI, –5.8 to 4.3]) during the application of 5 cm H₂O PEEP (P = 0.016) and during the application of 10 cm H₂O PEEP (–10.3 cm H₂O [95% CI, –15.2 to –5.4] vs. –2.8 cm H₂O [95% CI, –8.0 to 2.3]; P = 0.041; fig. 2A). No significant difference in absolute transdiaphragmatic pressure after 6 h of mechanical ventilation between the two groups was observed for the higher levels of PEEP (15 and 20 cm H₂O PEEP; fig. 2A).

The mean estimated transdiaphragmatic pressure decreased for each applied PEEP level in the restrictive group contrary to the liberal group, although the change was not statistically significant for PEEP 20 cm H₂O (fig. 2B). No differences in the transdiaphragmatic pressures were observed with the application of the same level of PEEP when applied in an increasing (PEEP 5-10-15-20 cm H₂O) or decreasing order (PEEP 20-15-10-5 cm H₂O; data not shown).

In two animals (restrictive group: n = 1; liberal group: n = 1), no microcirculation could be assessed due to technical problems with the video microscope. There was a significant decrease in the density of the present microvessels, expressed as the number of crossings, in animals receiving a restrictive fluid regimen (34.0 [25th to 75th percentile, 22.0 to 42.0]) compared to animals receiving liberal fluids (46.0 [25th to 75th percentile, 43.5 to 54.0]; P = 0.015). The difference in microvascular flow between the two groups was not significant (P = 0.176; fig. 3, A and B). Representative still images of microvascular flow are shown in fig. 3, C and D.

When studying the effect of diaphragm stimulation at various levels of PEEP, it was observed that increasing the PEEP level caused a decline in the generated transdiaphragmatic pressure. To establish the effect of the level of PEEP on the transdiaphragmatic pressures without the effect of induced pediatric ARDS or fluids, we used the baseline measurements of the entire group (restrictive and liberal group together) because in both groups, no difference in treatment existed at that time. High levels of PEEP caused a significant reduction in transdiaphragmatic pressures (fig. 4). For example, an acute increase in PEEP from 5 to 20 cm H₂O PEEP reduced transdiaphragmatic pressures by 27.3% (17.3 cm H₂O [95% CI, 14.0 to 20.5] vs. 12.6 cm H₂O [95% CI, 9.2 to 15.9]; P < 0.0001).

Standard hematoxylin and eosin staining did not show considerable endomysial edema or vacuolization of muscle fibers, especially not in the liberal group (fig. 5). As expected in this time window, no modification in the myosin heavy chain isoform expression profile occurred between the groups as the percentages of type 1 and type 2 muscle fibers were not different.

Quantification of fiber size in type 1 and type 2 fiber populations did not reveal significant differences between the groups. The median cross-sectional areas of slow-twitch (type 1) and fast-twitch fibers (type 2) in the liberal group were 470 µm² (interquartile range, 342 to 522) and 458 µm² (interquartile range, 309 to 500), respectively, and for the restrictive group, 498 µm² (interquartile range, 379 to 687) and 467 µm² (interquartile range, 368 to 574), respectively (fig. 6). The groups did not differ with respect to fiber proportion (%) and the numerical proportions or area fractions of slow-twitch (I) and fast-twitch fibers (fig. 6).

The diaphragmatic levels of proinflammatory cytokines interleukins 1β, 6, and 8 and tumor necrosis factor α, as well as the anti-inflammatory cytokine interleukin 10, were similar between the groups (fig. 7). Furthermore, no between-group differences in the concentration of malondialdehyde in diaphragm tissue were observed (fig. 7).

A restrictive fluid regimen causes a decline in diaphragmatic muscle force compared to a liberal fluid regimen. Contrary to our hypothesis, we observed that the use of a liberal fluid strategy did not lead to the formation of edema in or around the diaphragm muscle fibers in the early phase of experimental pediatric ARDS. Furthermore, we demonstrated that the acute application of PEEP decreases the force-generating capacity of the diaphragm in a dose-related way. These findings are novel and provide new insight into the mechanism of critical illness–associated diaphragm dysfunction and may have implications for the treatment of patients with pediatric ARDS in future.

After 6 h of mechanical ventilation, the force-generating capacity of the diaphragm was lower in the restrictive fluid group compared to the liberal fluid group, especially at lower PEEP. No histological explanation can be provided, as we found no differences, in particular those pertaining to necrosis in muscle fibers, between the two groups.

Furthermore, although previous studies have shown that proinflammatory cytokines and oxidative stress may impair the diaphragmatic contractility,^38,39  we observed similar concentrations of these mediators between the groups. Our results are comparable to previous studies comparing proinflammatory cytokines in the lung between a liberal fluid resuscitation strategy versus a restrictive fluid resuscitation strategy in animals, also showing similar levels of proinflammatory cytokines in both groups.^17,40,41 

The restrictive fluid group can be characterized as having a lower intravascular volume while being treated with relatively high dosage of vasopressors. A possible explanation for the reduced diaphragm force–generating capacity in the restrictive group may be the result of disturbances in the perfusion of the (capillary) blood vessel network as the capillary density of the diaphragm microcirculation was significantly lower in the restrictive group compared to the liberal group. The vasoconstrictive effect of norepinephrine in high dosages in a situation of relative hypovolemia might have led to a decrease in microvascular density, which ultimately may have resulted in decreased diaphragmatic tissue oxygenation and force-generating capacity of the diaphragm.

Another explanation for the reduced diaphragm force–generating capacity in the restrictive group could also be the (additional) result of disturbances at the level of the neuromuscular junction. In experimental studies on isolated phrenic-diaphragm preparation of mice, it was demonstrated that norepinephrine may decrease the frequency of spontaneous quantal release of acetylcholine and increase the degree of asynchrony of acetylcholine secretion.^42,43  This might negatively affect the force-generating capacity of the diaphragm and is in line with our findings. However, research on this topic was beyond the scope of the current study.

Contrary to our hypothesis, liberal fluids in the early phase of pediatric ARDS did not lead to the formation of endomysial edema or swelling of the diaphragm muscle fibers as the endomysial space and the mean cross-sectional areas between the two groups were comparable. Myofibrillar and endomysial edema have been demonstrated in animal septic shock and ischemic models.^14,15  However, the causal mechanism, such as septic shock and ischemia, responsible for the formation of edema was different in the previously mentioned studies. Therefore, it is possible that the formation of edema in and around the diaphragm muscle fibers during fluid overload may not occur or develop later during the course of the disease.

In the early stage of pediatric ARDS, the acute application of PEEP decreases the in vivo force-generating capacity of the diaphragm in a dose-related effect; the higher the PEEP, the greater the decrease in transdiaphragmatic pressure. To our knowledge, no studies have explored this dose-related effect of PEEP on the transdiaphragmatic pressures in vivo in an animal model of pediatric ARDS.

A possible explanation for the decrease in transdiaphragmatic pressure as a result of the application of PEEP is that the acute application of PEEP causes a caudal movement of the diaphragm dome, causing fiber and sarcomere reduction in the zone of apposition, as has been shown in vitro in rat studies.³⁴  In this way, the diaphragm muscle fibers are forced to contract at a shorter length, leading to a reduction in the force-generating capacity of the diaphragm.³⁴  As an explanation for the dose-related effect, we postulate that the reduction in sarcomere length becomes more pronounced with the higher levels of PEEP. Second, as recently shown, mechanical ventilation increases vascular resistance and impairs diaphragm perfusion, an effect more pronounced with higher PEEP levels.⁴⁴  Whether the decrease in diaphragm perfusion indeed causes lower transdiaphragmatic pressures in our model warrants further investigation.

No differences in the diaphragm function were found between the two groups with the application of 20 cm H₂O PEEP. A possible explanation might be that with this high level of PEEP, the diaphragm behaves as a widening piston,⁴⁵  forcing the muscle fibers to act at a suboptimal length, generating low force. The impact of such a high level of PEEP is tremendous on the diaphragm force–generating capacity, leaving no additional effect for other negative factors.

Some limitations of our study should be acknowledged. First, complete blinding after allocation of the groups was not possible for the research group as they were responsible for the assigned treatment. However, biopsy handling and scoring of the microvascular images was blinded for the researcher. Second, we observed that the use of a liberal fluid strategy did not lead to the formation of edema in the diaphragm in the early phase of experimental pediatric ARDS. Whether edema of the diaphragm develops beyond the time frame of the study remains to be investigated.

Third, as no functional measurements (force generations and microvasculature) were performed on nonrespiratory skeletal muscles, it is unclear whether our findings are specific to the diaphragm muscle.

Fourth, we expected that animals in the liberal group would need higher levels of PEEP due to the development of compression atelectasis, subsequently leading to a change in the end-expiratory lung volume. These changes may influence the position of the diaphragm at the zone of apposition, subsequently affecting the force-generating capacity of the diaphragm.³⁴  However, at the end of the experiment, no significant difference in transpulmonary pressure at end exhalation, lung compliance, chest wall compliance, and driving pressure between the two groups existed, suggesting that no change in the end-expiratory lung volume occurred. This means we may not have had to perform diaphragm stimulations under different levels of PEEP. However, by performing the diaphragm stimulations under different levels of PEEP, we have now gained additional insight into the acute effects of PEEP in vivo on the diaphragm function. However, further studies are needed to gain insight in the long-term effects of different levels of PEEP on diaphragm function.

Fifth, due to technical reasons, it was not possible to measure the transdiaphragmatic pressure during all different levels of PEEP in all animals.

Sixth, our experiment was performed in female lambs only. Studies have shown that testosterone might increase diaphragm contractility in rats.^46,47  Given the young age of the lambs, we consider changes in the level of testosterone to be small between the two sexes, and it is unlikely that the female predominance has influenced our results. In addition, there are no data to support the possibility that the effects of the oleic acid or resuscitation strategy are sex-dependent.

Finally, as this is an experimental animal model, the results cannot directly be translated to clinical strategies but emphasize the importance of future studies in humans.

The strength of our study was that we performed this experiment under controlled and uniform conditions with standardized, validated, and direct measurements in which we included in vivo function, perfusion, and histology of the diaphragm. Furthermore, as our study was part of a more extensive experiment with the same research design but with different research questions (focused on the cardiopulmonary effects of fluid strategy during pediatric ARDS),¹⁷  we were able to minimize the number of animals used.

Critical illness–associated diaphragm weakness develops in the majority of mechanically ventilated critically ill patients, may be associated with prolonged duration of mechanical ventilation, and may be associated with mortality.^1–5  The current study shows that in the early phase of severe experimental pediatric ARDS, a restrictive fluid strategy may adversely affect diaphragm force–generating capacity. Although the exact mechanism has yet to be elucidated, our findings are novel and hypothesis-generating for future clinical studies. In addition, our study underscores that the optimal modalities of fluid management and PEEP titration in patients with ARDS are challenging and that the effects appear to be organ-specific. In other words, a lung-protective strategy characterized by high PEEP and early fluid restriction may have detrimental effects on other organs, including the respiratory muscles.

These new mechanisms may help identify patients at risk for diaphragm dysfunction. The ultimate goal is an individualized integrated lung and diaphragm-protective approach for the management of pediatric ARDS.

Early fluid restriction decreases the force-generating capacity of the diaphragm in the acute phase of experimental pediatric ARDS. In addition, the application of PEEP itself decreases the force-generating capacity of the diaphragm in a dose-related way, which was more pronounced in a restrictive regimen compared to a liberal fluid regimen. These observations provide new insights into the mechanism of critical illness–associated diaphragm dysfunction.

The authors thank Alex Hanssen, Ing. (Animal Research Facility, Radboud University Nijmegen, The Netherlands), for his excellent technical assistance during the experiment. They also express their gratitude to Theo G. M. Hafmans, BsC., Paul H. K. Jap, M.D., Ph.D. (Department of Biochemistry, Radboud University Medical Centre, Nijmegen, The Netherlands), and Jelle Gerretsen, BsC., (Department of Intensive Care Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands) for their extensive effort in the histological preparations and biochemical analysis. Medtronic Inc. (Maastricht, The Netherlands) and Maquet (Hilversum, The Netherlands) provided the equipment for the external stimulator (models 990066, 19038, 37022, and 8840) and the mechanical ventilator (Servo-i), respectively.

Support was provided solely from institutional and/or departmental sources.

Dr. Heunks has received funding from Getinge (Gothenburg, Sweden), Fisher and Paykel (Tilburg, The Netherlands), and Liberate Medical (Crestwood, Kentucky). The other authors declare no competing interests.