BACKGROUND

Delivery of tidal volume by mechanical ventilation to a deeply sedated patient does not follow normal physiological transpulmonary pressure gradients as there are no associated respiratory muscle contractions (1–3). In the supine position, tidal volume is distributed more to non-dependent, ventral alveoli, causing overdistention, and alveolar collapse is promoted in dependent, dorsal alveoli as they receive less volume, resulting in atelectasis (1, 4). Atelectasis causes injury to lung tissue through the shearing forces created during cycles of alveolar collapse and expansion during mechanical ventilation (5). Atelectasis is further encouraged by the use of low-volume, lung-protective ventilation strategies, gravitational forces, and increased hydrostatic pressure from abdominal contents due to patient position (6–11). Increased atelectasis-related pulmonary shunt exacerbates hypoxia and hypercarbia, which has been shown to be a potent inducer of inflammation and lung epithelial injury (8, 12). The proportion of tidal volume is increased in open alveoli when atelectasis forms, which increases respiratory system driving pressure, leading to worsening overdistension and injury (5, 13–16). Decreased driving pressure is strongly associated with increased survival in patients with acute respiratory distress syndrome (ARDS) (17).

Many critically ill, mechanically ventilated patients are administered deep sedation to either facilitate achieving therapeutic goals, or to prevent asynchronous interaction with the mechanical ventilator (18, 19). Prolonged deep sedation worsens ventilator-induced lung injury (VILI) and puts the patient at risk for ventilator-induced diaphragm atrophy (20–23). The PLUG group has identified a need to combine both lung- and diaphragm-protective ventilation in a single strategy (24).

Concepts from the field of functional electrical nerve stimulation provide a mechanism to combat both atelectasis-driven lung injury and diaphragm atrophy in sedated patients unable to breathe spontaneously, by delivering a targeted stimulus to the phrenic nerves (25–28). This neurostimulation contracts the diaphragm, preserving muscle strength and counteracting the compressive pressure from abdominal contents (25, 28). It also promotes more physiologically normal distribution of tidal volume, which helps to keep dorsal alveoli open as they receive more tidal volume (25). Electrically stimulated diaphragmatic activity that prevented atelectasis was shown in a series of 12 patients undergoing intraabdominal surgery who received right-sided percutaneous electrical stimulation of the phrenic nerve in the neck (25). A 9.5 French central venous catheter equipped with stimulation electrodes has been developed recently (LIVE Catheter, Lungpacer Medical Inc.) (29). The catheter is temporarily and percutaneously inserted in the left subclavian vein, and stimulation current is delivered transvenously to capture the phrenic nerves for diaphragm activation. This activation is delivered adjunctively and, in synchrony with, traditional mechanical ventilation. This ventilatory adjunct has been shown to mitigate ventilator-induced diaphragm atrophy and dysfunction in a pig model and has potential for preventing compressive atelectasis and atelectrauma (28). It has been shown to effectively elicit diaphragm contractions in humans and is presently under evaluation in clinical trials (ClinicalTrials.gov: NCT03783884) (27). Diaphragm neurostimulation offers a potential solution for a protective ventilation strategy that protects both the diaphragm and the lungs at the same time, but it is not self-evident that elicited respiratory efforts are similar to diaphragm contractions during spontaneous breaths. Therefore, it is imperative to carefully study this promising intervention to ensure that it provides some of the benefits associated with the diaphragm contractions that occur during normal physiological spontaneous breathing.

The purpose of this study was to investigate the effects of phrenic nerve paced diaphragm contractions on atelectasis formation and alveolar homogeneity in a preclinical model, which may apply to heavily sedated patients receiving positive-pressure ventilation. We aim to show that TTDN can restore some of the benefits of diaphragm contraction to preclinical subjects that are deeply sedated, thereby offering a potential mechanism to prevent the development of all the aspects that contribute to VILI and VIDD during the acute sedation phase of their intensive care unit (ICU) course in humans. Our primary hypothesis is that temporary, transvenous diaphragm neurostimulation (TTDN), in combination with mechanical ventilation, will restore a more physiological distribution of tidal volume and reduce end-expiratory lung volume loss. This will improve $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ and reduce the amount of atelectasis formation, reducing alveolar inhomogeneity, compared to mechanical ventilation alone.

METHODS

Twenty-five healthy female Yorkshire pigs (43–71 kg) underwent 50 h of ventilation in ICU conditions. One group received volume-control mechanical ventilation only (MV group, n = 10). A second group received synchronous TTDN during volume-control ventilation on every second breath (MV + TTDN50% group, n = 8). A third group received synchronous TTDN during volume-control ventilation on every breath (MV + TTDN100% group, n = 7). All animals were ventilated in the supine position. Six pigs, never intubated or ventilated, served as histology controls (NV group, n = 6). The study was conducted in accordance with Canadian Council for Animal Care guidelines and approved by the University of British Columbia and Simon Fraser University Animal Ethics Committees. Detailed descriptions of protocols are provided in the Online Data Supplement (see https://doi.org/10.6084/m9.figshare.14077430). Animals were weighed at the start of the experiment, and animal length was measured from the tip of the snout to the base of the tail.

Both of the MV + TTDN groups received a neurostimulation catheter inserted percutaneously into the left subclavian vein. TTDN was delivered in synchrony with the inspiratory phase (Ti) of ventilator-delivered breaths as previously published (28). Synchrony was achieved using a flow sensor inline at the ventilator circuit-endotracheal tube junction. Stimulation was initiated when the start of inspiration was detected. The duration of stimulation was set to be the same length as the ventilator’s Ti. TTDN was delivered at 25 Hz with a stimulation intensity level set to target a reduction in ventilator (Paw) pressure-time product (PTP) of 15%–20% when compared to MV-only PTP. Refer to Fig. E2 in the Online Data Supplement for an example of PTP reduction. MV and MV + TTDN groups were deeply sedated with intravenous anesthesia to ablate respiratory drive and were monitored with Bispectral Index Monitoring, validated for use in pigs, to measure the level of sedation under anesthesia (30). Neuromuscular blocking agent was not used in this study as this blocks nerve conduction to the diaphragm, which precludes diaphragm neurostimulation. We have chosen this model in order to investigate whether the positive effects of diaphragm contractions during spontaneous breathing can be restored to subjects who cannot breathe spontaneously due to a treatment requirement for deep sedation. NV animals were lightly sedated briefly with inhaled anesthetic via mask to facilitate gathering relevant data, before euthanasia.

The MV and MV + TTDN groups were ventilated using a lung-protective strategy with tidal volume of 8 mL/kg in volume control, PEEP of 5 cmH₂O and $\text{F}{\text{I}}_{{\text{O}}_2}$ titrated to $\mathrm{S}{\mathrm{p}}_{{\mathrm{O}}_2}$ > 94% (18). We used volume-control and not pressure-control because the combination of TTDN with pressure-control ventilation would have resulted in large variations in tidal volume, preventing a consistent lung-protective ventilation protocol based on tidal volume. Lung mechanics were measured every 2 h by electrical impedance tomography (EIT; Dräger PulmoVista 500). The EIT band was placed at the 6th intercostal space and was left in place for the duration of the experiment. Esophageal balloons were used to measure transpulmonary pressures in all subjects. Transpulmonary pressures were measured for both MV and MV + TTDN breaths at end-inspiration and end-expiration. Inflammatory cytokines were obtained from the right cranial lobe via bronchoscopy. This is the only lobe accessible with commercially available bronchoscopes due to the length of the trachea in pigs of this size. At necropsy, lung tissue samples were taken from the left lung only, at five specific locations (Fig. 3 details sample locations) and fixed in formalin and embedded in paraffin. Lungs were not reinflated to standard transpulmonary pressure as this posed a distinct threat of re-recruitment of atelectatic alveoli. Alveolar expansion at the time of tissue fixation was quantified by measuring alveolar chord length (31). Refer to the Online Data Supplement for detailed protocols.

A power calculation with an alpha of 0.05 and a beta of 0.80 based on the $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ ratio changes seen in the experiment by Grasso et al. with negative-pressure ventilation in lung-injured rabbits (350 mmHg vs. 75 mmHg, SD 75) yields two animals per arm (32). A minimum of six animals per group was used to ensure that groups were large enough to be able to use nonparametric statistical tests. Additional animals were added to the MV, MV + TTDN50% and MV + TTDN100% groups to replace lost tissue samples from a freezer failure, procedural challenges during the 2020 COVID-19 pandemic, and as part of an effort to balance experimental pairs. Statistical analysis was performed using GraphPad Prism version 8.0.0 (GraphPad Software, San Diego, CA; www.graphpad.com). All measurements are reported as median (IQR), 95% confidence interval of the median and were tested using nonparametric statistical tests. Medians among all groups were compared using Kruskal–Wallis one-way analysis of variance. A post hoc Dunn’s test of multiple comparisons was used to test which specific groups were different from each other if Kruskal–Wallis analysis showed a significant result. As repeated-measures ANOVA cannot handle missing values, we analyzed the data by fitting a mixed model as implemented in GraphPad Prism 8.0. This mixed model uses a compound symmetry covariance matrix and is fit using restricted maximum likelihood (REML). Fixed effects were time, TTDN dose, and TTDN dose interaction with time. Random effect was subjects. A Geisser–Greenhouse correction was used to correct for lack of sphericity. Longitudinal data are graphed as median (IQR) with simple linear regression for line of best fit.

RESULTS

Alterations in Distribution of Tidal Volume by TTDN

Ventilator-triggered TTDN diaphragm contractions in synchrony with the inspiratory phase of a volume-control breath changed the distribution of tidal volume. Dorsal distribution of tidal volume was different between the groups, P < 0.0001 (Table 3, Fig. 1A). Tidal volume in the ventral lung region was also different between all groups, P < 0.0001 (Table 3, Fig. 1A). Dorsal tidal volume changed from 47% (45–53) for breaths with no diaphragm contraction (passive) to 53% (50–57) for breaths when the diaphragm was contracted (active) in the same animal of the MV + TTDN50% group, with a concurrent shift in ventral tidal volume, 53% (47–55) to 47% (44–51) in the same animal of the MV + TTDN50% group, P = 0.0499. Figure 1B shows an example of an EIT image documenting the redistribution of tidal volume from ventral to dorsal lung regions between passive and active breaths in the same animal.

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Table 3. Distribution of tidal volume in dorsal and ventral lung regions

Tidal volume distribution	Parameter	Dorsal tidal Volume	Ventral Tidal Volume
	Units	%	%
NV	Median	61	39
	IQR	49–70	30–51
	95% CI	48–73	27–52
MV	Median	37	63
	IQR	32–45	55–68
	95% CI	15–46	54–85
MV + TTDN50% (Passive)	Median	47	53
	IQR	45–53	47–55
	95% CI	44–54	45–56
MV + TTDN50% (Active)	Median	53	47
	IQR	50–57	44–51
	95% CI	48–68	32–52
MV + TTDN100%	Median	59	41
	IQR	57–70	30–43
	95% CI	53–77	23–48
Kruskal–Wallis Test (P value)		<0.0001	<0.0001
Dunn's Multiple Comparisons Test (P value)
NV vs. MV		0.0029	0.0029
NV vs. MV + TTDN50% Passive		ns	ns
NV vs. MV + TTDN50% Active		ns	ns
NV vs. MV + TTDN100%		ns	ns
MV vs. MV + TTDN50% Passive		ns	ns
MV vs. MV + TTDN50% Active		0.0182	0.0168
MV vs. MV + TTDN100%		0.0029	<0.0001
MV + TTDN50% Passive vs. MV + TTDN100%		ns	ns
MV + TTDN50% Active vs. MV + TTDN100%		ns	ns

“–” = not applicable. MV, mechanical ventilation; ns, not significant; TTDN, transvenous diaphragm neurostimulation.

Change in End-Expiratory Lung Volume (as Measured by EIT), a Trend of Atelectasis

Net end-expiratory lung volume (EELV) loss at the end of 50 h was different between the three ventilated groups [1203 mL (804–1554) MV group, 1038 mL (677–1203) MV + TTDN50% group and 677 mL (398–985) MV + TTDN100% group, P = 0.0034, Table 4]. The mixed-effect analysis shows that EELV was affected by time (P < 0.0001) and TTDN dose (P = 0.0043) and that the interaction of TTDN dose with time was also significant, P = 0.0034 (Fig. 2A). Figure 2B is an example of EIT analysis showing the loss of EELV (orange) and the gain (blue) over 50 h for each group.

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Table 4. Net loss of end-expiratory lung volume as measured by EIT, at the end of 50 h of mechanical ventilation

Net Loss of End-expiratory Lung Volume	Parameter	dEELV Global
	Units	Ml
MV	Median	1,203
	IQR	985–1554
	95% CI	984–1638
MV + TTDN50%	Median	1,038
	IQR	845–1259
	95% CI	461–1423
MV + TTDN100%	Median	677
	IQR	398–804
	95% CI	372–848
Kruskal–Wallis Test (P value)		0.0034
Dunn's Multiple Comparisons Test (P value)
MV vs. MV + TTDN50%		ns
MV vs. MV + TTDN100%		0.0028
MV + TTDN50% vs. MV + TTDN100%		ns
Mixed-effects Analysis (P value)	Time	<0.0001
	Group	0.0043
	Interaction	0.0034

“–” = not applicable. MV, mechanical ventilation; ns, not significant; TTDN, transvenous diaphragm neurostimulation.

Alveolar Expansion and Tissue Homogeneity

The alveolar chord length is different between all groups for each tissue sample location (P < 0.0001 location 1, P = 0.0045 location 2, P < 0.0001 location 3, P < 0.0001 location 4, P < 0.0001 location 5). Samples taken from the lower lobe (sample locations 1–3) were not different between the NV and MV + TTDN100% group in the post hoc analysis. Referring to Table 5, Figure 3 shows hematoxylin and eosin-stained sections of lung tissue from each sample location and group.

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Table 5. Alveolar chord length for each tissue sample location

Alveolar Chord Length Measurement	Parameter	Sample Location 5	Sample Location 4	Sample Location 3	Sample Location 2	Sample Location 1
	Units	µm	µm	µm	µm	µm
NV	Median	83.99	82.71	86.54	80.28	84.66
	IQR	59.10–117.90	56.67–120.30	59.95–123.60	53.64–118.00	57.67–119.00
	95% CI	81.04–86.72	80.18–85.79	83.51–89.82	76.36–84.78	81.44–87.01
MV	Median	109.6	108.4	93.51	78.95	57.85
	IQR	77.69–163.60	76.68–166.80	63.90–142.40	55.84–122.10	46.41–76.02
	95% CI	104.90–113.70	105.30–114.20	89.47–96.19	77.03–81..36	53.82–60.54
MV + TTDN50%	Median	92.04	97.67	73.7	76.73	73.81
	IQR	64.63–138.90	64.45–150.10	52.38–112.90	51.07–109.00	50.37–105.60
	95% CI	88.57–95.65	94.07–101.70	71.45–76.37	74.91–79.39	69.90–77.19
MV + TTDN100%	Median	85.94	88.12	80.72	76.68	74.67
	IQR	64.58–118.10	67.60–123.80	58.53–117.00	59.20–108.3	55.84–104.30
	95% CI	84.08–88.12	84.78–90.81	82.74–78.70	74.67–78.71	71.98–78.03
Kruskal–Wallis Test (P value)		<0.0001	<0.0001	<0.0001	0.0045	<0.0001
Dunn's Multiple Comparisons Test (P value)
NV vs. MV		<0.0001	<0.0001	0.0001	ns	<0.0001
NV vs. MV + TTDN50%		<0.0001	<0.0001	<0.0001	ns	<0.0001
NV vs. MV + TTDN100%		0.0133	<0.0001	ns	ns	ns
MV vs. MV + TTDN50%		<0.0001	<0.0001	<0.0001	0.0095	<0.0001
MV vs. MV + TTDN100%		<0.0001	<0.0001	<0.0001	ns	<0.0001
MV + TTDN50% vs. MV + TTDN100%		0.0082	0.0023	<0.0001	0.0055	ns

“–” = not applicable. MV, mechanical ventilation; ns, not significant; TTDN, transvenous diaphragm neurostimulation.

Ventilation Pressures and Dynamic Compliance

Esophageal pressure measured at end-inspiration was not different at baseline or study end, between groups or for the interaction of TTDN dose with time. Esophageal pressure was affected by time, P = 0.0214 (Table 6). Plateau pressure was different between the three groups at study end, P = 0.0258. The mixed-effect analysis shows that plateau pressure was affected by time (P = 0.0054) and TTDN dose (P = 0.0203). The interaction of TTDN dose with time was not significant. Driving pressure (plateau pressure minus PEEP) was different at study end between the three groups, P = 0.0258. The mixed-effect analysis shows that driving pressure was affected by time (P = 0.0118) and TTDN dose (P = 0.0251). The interaction of TTDN dose with time was not significant. Transpulmonary plateau pressure was different between all three groups at both baseline (P = 0.0015) and study end (P = 0.0015). The mixed-effect analysis shows that transpulmonary plateau pressure was affected by time (P = 0.0189) and TTDN dose (P = 0.0016). The interaction of TTDN dose with time was not significant. Referring to Table 6 and Fig. 4, measurements were taken for all breaths (MV or MV + TTDN as dictated by the study group).

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Table 6. Esophageal pressures, ventilation pressures, compliance, and $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ ratio at baseline and at the end of 50 h of mechanical ventilation

Pressures, Compliance and P/F Ratio		Esophageal Pressure			Plateau Pressure			Driving Pressure			Transpulmonary Plateau Pressure			Dynamic Compliance			Static Compliance			$\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ Ratio
		cmH2O			cmH2O			cmH2O			cmH2O			ml/cmH2O			ml/cmH2O			–
		Median	IQR	95% CI	Median	IQR	95% CI	Median	IQR	95% CI	Median	IQR	95% CI	Median	IQR	95% CI	Median	IQR	95% CI	Median	IQR	95% CI
MV	Baseline	9	6–14	6–15	16	14–16	14–17	11	9–11	9–12	9	8–10	7–11	39	36–41	36–41	44	38–48	37–52	521	455–552	450–559
	Study End	7	6–10	5–10	19	18–21	18–22	14	13–16	13–17	14	13–16	11–17	48	33–53	33–53	30	29–36	27–36	403	358–444	327–457
MV + TTDN50%	Baseline	9	6–13	5–15	16	15–18	13–25	11	11–13	8–20	10	8–13	5–14	39	35–47	34–61	35	33–43	28–50	462	438–498	420–547
	Study End	7	6–10	4–10	20	18–22	17–24	15	13–17	12–19	14	12–16	10–17	30	24–40	13–42	30	27–35	24–38	374	336–413	72–497
MV + TTDN100%	Baseline	10	8–12	8–13	16	15–17	14–17	12	11–12	10–12	6	5–7	5–8	69	55–79	49–110	46	44–47	44–48	492	468–509	420–512
	Study End	10	6–12	4–13	18	16–18	16–19	13	12–14	10–16	7	5–13	5–15	67	62–79	49–81	42	37–44	34–55	433	430–470	417–540
Kruskal–Wallis Test (P value)	Baseline	ns			ns			ns			0.0015			0.0006			0.0257			ns
	Study End	ns			0.0258			0.0258			0.0018			<0.0001			0.0035			0.0440
Dunn’s Multiple Comparisons Test (P value)
MV vs. MV + TTDN50%	Baseline	–			–			–			ns			ns			ns			–
MV vs. MV + TTDN100%		–			–			–			0.0113			0.0044			ns			–
MV + TTDN50% vs. MV + TTDN100%		–			–			–			0.0079			0.0189			0.0257			–
MV vs. MV + TTDN50%	Study End	–			ns			ns			ns			ns			ns			ns
MV vs. MV + TTDN100%		–			ns			ns			0.0232			ns			0.0138			ns
MV + TTDN50% vs. MV + TTDN100%		–			0.0431			0.0431			0.0367			0.0006			0.0061			0.0412
Mixed-effects Analysis (P value)	Time	0.0214			0.0054			0.0118			0.0189			ns			ns			<0.0001
	Group	ns			0.0203			0.0251			0.0016			<0.0001			<0.0001			0.0393
	Interaction	ns			ns			ns			ns			0.0048			ns			ns

“–” = not applicable. MV, mechanical ventilation; ns, not significant; TTDN, transvenous diaphragm neurostimulation.

EIT-measured dynamic compliance was different between all three groups at both baseline (P = 0.0006) and study end (P < 0.0001). The mixed-effect analysis shows that dynamic compliance was not affected by time, but the effect of TTDN dose was significant (P < 0.0001). The interaction of TTDN and time was also significant, P = 0.0048. The slope of the linear regression was different between all three groups (P < 0.0001). Static compliance was different between the three groups at both baseline (P = 0.0257) and study end (P = 0.0035). The mixed-effect analysis shows that static compliance was not affected by time, but the effect of TTDN dose was significant (P < 0.0001). The interaction of TTDN dose and time was not significant. Measurements were taken for all breaths (MV or MV + TTDN as dictated by the study group) (Table 6 and Fig. 4).

Change in $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$

The mixed-effect analysis shows that $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ was affected by time (P < 0.0001) and TTDN dose (P = 0.0393). The interaction of TTDN dose and time was not significant (Table 6). The longitudinal change in $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ over 50 h was not different; however, the slope of the linear regression was different between all three groups (P = 0.0044; Fig. 5).

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DISCUSSION

TTDN combined with lung-protective mechanical ventilation distributed tidal volume in a more physiological pattern, mitigated the loss of EELV, improved alveolar homogeneity, and preserved $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ when provided on every breath. These results indicate that TTDN on every breath protects the lungs against potential ventilator-induced lung injury by keeping them “open.” Improved lung compliance and the reduction of both transpulmonary plateau and driving pressures support the biological plausibility and experimental validity of this work. The positive findings in this study, as well as the absence of negative interactions such as asynchrony, attest that it is reasonable to evaluate the impact of TTDN in human patients.

The significant mitigation of end-expiratory lung-volume loss with the use of adjunctive TTDN on every breath is an important finding and establishes a biological foundation for the potential clinical benefit of TTDN in critically ill patients. There was twice as much end-expiratory lung volume lost in the MV group compared to the MV + TTDN100% group, with the MV + TTDN50% group being closer to the MV group. This demonstrates that TTDN on every breath preserves more end-expiratory lung volume than it does on every second breath, and thus, the dose of TTDN is important. The more alveoli that are preserved for tidal respiration, the greater the reduction in the overall stress on individual units, thereby reducing atelectrauma and cyclic strain. This is further supported by the improved $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$, lower transpulmonary plateau pressure, and respiratory system driving pressures in the MV + TTDN100% group versus the MV + TTDN50% group. TTDN on every breath reduced driving pressure in comparison with the gold-standard lung-protective ventilation strategy alone. We have previously shown that TTDN on every second breath, targeting the same ventilator PTP reduction as this study, mitigates ventilator-induced diaphragm atrophy (28). This combined with the potential for the reduction of atelectrauma, when delivered on every breath, suggests that diaphragm neurostimulation may be a promising candidate for a combined lung- and diaphragm-protective ventilation strategy.

Lung inhomogeneity results in abrupt changes in the configuration and internal pressures of alveoli (5). Increased inhomogeneity leads to stress amplification in the walls of the neighboring lung units (5, 33). Atelectasis increases lung inhomogeneity, causing an up to fourfold increase in stress and strain in the areas adjacent to areas of collapse, driving lung injury (5, 33). Once injury is initiated in an alveolus, less stress and strain are required to further damage it, described as the two-hit hypothesis (33, 34). TTDN preserves the range of alveolar expansion when delivered both on every breath and every second breath; however, the MV + TTDN100% group had a range closer to the NV group, with the lower lobe (location 1–3) not different in post hoc analysis. The MV group showed the largest loss of end-expiratory lung volume and the largest range of alveolar chord length and thus alveolar inhomogeneity (57.85–109.60 µm). The smallest measurements in the MV group were in the base of the lower lobe (sample site 1), the area with the most atelectasis, whereas the largest were in the upper regions of the upper lobe, the area most prone to overdistension and injury. The MV + TTDN100% group was the most similar to the NV group (74.67–88.12 µm vs. 80.28–86.54 µm), having the least inhomogeneity of the ventilated groups and the lowest loss of end-expiratory lung volume. The impact of reduced lung compliance on alveolar chord length cannot be excluded; however, the MV and MV + TTDN50% groups had similar compliance, but their alveolar chord length ranges were different. These alveolar chord length findings are supported by the pattern of EELV loss, alterations in driving pressure, and gross histology observation. The reduction in inhomogeneity by TTDN is a key finding of this study as it elucidates how this emerging intervention may encourage alveolar homogeneity while preserving end-expiratory lung volume thereby promoting an “open lung.”

$\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$, an indicator of pulmonary shunt, is well correlated with atelectasis (35–37). While not reaching the level of mild lung injury (<300) in any group, $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ was better preserved in the MV + TTDN100% group compared to the other groups. The MV + TTDN50% group had two out of eight animals experience significant mucus plugging episodes in the last 8 h of the experiment that reduced $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ ratios to a 95% CI of 72–497 for study end, whilst remaining clinically stable. This phenomenon has also been described in a case report from a human clinical trial using this technology for diaphragm rehabilitation (38). This did not occur in the group receiving TTDN on every breath and is possibly related to the intermittent pattern of diaphragm contractions being more effective at mobilizing mucus plugs in the same way as a cough-assist procedure (39). Although lung-protective ventilation over 50 h did not have a serious impact on $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ in this healthy lung model, TTDN on every breath maintained an “open lung,” leading to more alveoli participating in gas exchange, thereby leading to better $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ over time. This was likely not pronounced due to the significant physiological reserves in a healthy lung.

There was a systematic reduction in the transpulmonary plateau pressure readings in the MV + TTDN100% group over time. This pattern is echoed in both the dynamic and static compliance measurements, suggesting that TTDN on every breath changes compliance in ways other than recruitment of collapsed alveoli, as it is evident at the start of the experiment. Pellegrini et al. have postulated that the diaphragm acts like a “brake” during expiration in spontaneous breathing by maintaining diaphragm tonicity even after a phasic contraction (40). The diaphragm thus regulates the transmission of hydrostatic pressure between the abdomen and the chest (40). They describe that a decrease in end-expiratory lung volume is associated with an electromechanical coupling that slows down the decrease of the phasic electrical diaphragm activity during expiration (40). They also demonstrated a residue of inspiratory electrical diaphragm activity that lasts for some time during inspiration (40). Lower end-expiratory lung volumes resulted in an increased residual diaphragm electrical activity for longer periods during expiration (40). Perhaps the changes in transpulmonary pressure and dynamic compliance seen in the MV + TTDN100% group are due to reinstating this diaphragm tonicity in expiration that reduces the transmission of abdominal pressure to the lungs. TTDN on every second breath does not have the same effects on transpulmonary plateau pressures and dynamic compliance, suggesting that the dose of diaphragm stimulation may be important in this phenomenon. Due to limitations in equipment acquisition, we have transdiaphragmatic pressure measurements for the MV + TTDN100% group only and thus cannot explore this theory in this data set.

Uncontrolled changes in tidal volume, pendelluft, increased lung perfusion, and patient-ventilator asynchrony may result from uncontrolled spontaneous patient efforts while on a controlled mode of mechanical ventilation and are hypothesized to have an adverse effect on lung injury (19, 41–43). TTDN may be seen to mimic these efforts as the diaphragm is contracting during a ventilator-delivered breath. However, a TTDN contraction is completely synchronous as it follows the ventilator during inspiration and the strength of the contraction is adjusted to reach a targeted PTP reduction (28). This study demonstrated no evidence of increased injury due to TTDN as lung injury scores were the same for all groups and inflammatory markers were not significantly different for any group. Extravascular lung water was also not significantly different, and therefore, TTDN did not cause an adverse transcapillary pressure gradient. This is relevant as strong respiratory efforts may lead to an increase in extravascular lung water such as is the case with negative-pressure pulmonary edema. Variation in tidal volume was prevented by the use of volume-controlled breaths, and there was no observed ventilator asynchrony or reverse triggering. The lack of ventilator asynchrony and absence of increased signs of lung injury in the TTDN groups support that TTDN does not increase the risk of lung injury and that it is reasonable to investigate the potential benefits of TTDN in patients with acute lung injury.

Our study has certain limitations, as it uses a preclinical model with healthy lungs and only a small number of subjects. We recognize that many ICU patients do not fit this model, but the patients who require extended sedation and mechanical ventilation, and are the most at risk for VIDD, do fit this model, and we have previously established that TTDN can offer protection from VIDD in healthy pigs (28). The goal of this study was to investigate the effects that TTDN can have on ventilator-induced lung injury, the results of which may apply to heavily sedated, critically ill patients receiving controlled mechanical ventilation. Whether the results are the same in human ICU subjects who are not healthy remains to be seen. This study was limited in its ability to measure transpulmonary driving pressure and transdiaphragmatic pressure as we did not obtain a full data set due to late equipment acquisition. Although pressure and compliance data are encouraging, more transpulmonary and transdiaphragmatic pressure data are needed to be conclusive. Critically ill patients that are affected by VILI are typically ventilated for more than 50 h; however, a longer duration of this experiment would have been technically difficult. The ability to do caudal lobe bronchial washings was inhibited by the length of bronchoscopes available, and thus, we could not fully investigate the level of lung inflammation and possible injury. Future investigation would benefit from using an injured-lung model to adequately evaluate the effect of TTDN on VILI in a nonhomogeneous, injured lung, to better reflect commonly encountered clinical scenarios. Although the MV + TTDN100% group subjects were heavier than the other groups, their bodies were proportionally larger and not just heavier, as the length-to-weight ratio was the same in all groups. We normalized tidal volume to 8 mL/kg.

Not only is TTDN beneficial but the manner in which it is delivered provides a specific benefit beyond simply a dose-dependent improvement. The MV + TTDN50% group received neurostimulated diaphragm contractions on every second breath, creating an environment where alveoli were constantly subjected to fluctuating stress and strain between active and passive breaths. The distribution of tidal volume in both the active and passive breaths in this group suggests that a single diaphragm contraction after a passive breath does not fully restore physiological tidal distribution. This, along with variable stress, may potentiate the positive effects seen from TTDN provided with every breath. Our previous work demonstrated a clear benefit to the diaphragm when TTDN was offered every second breath and this study shows that the benefit to the lungs is greater when TTDN is provided on every breath. Work is underway to evaluate the effect on diaphragm atrophy in this group. This will help to elucidate whether TTDN on every breath is the best candidate for a lung- and diaphragm-protective ventilation protocol, informing future experimental directions.

This study is important with findings that will inform the application of a promising new therapeutic modality. Our findings show that TTDN on every breath preserved $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ and required lower driving pressure than the reference-standard lung-protective mechanical ventilation strategy over 50 h in healthy lungs. There was less end-expiratory lung volume loss and better alveolar homogeneity with TTDN on every breath. Furthermore, TTDN resulted in reduced atelectasis and the accompanying potential for lung injury. This offers insights into the impact that atelectrauma has on VILI in noninjured lungs and its associated alveolar inhomogeneity. This study supports the hypothesis that synchronous diaphragm contraction on every breath during controlled mechanical ventilation is beneficial and is no more harmful than stand-alone, lung-protective mechanical ventilation in healthy, noninjured lungs. TTDN may offer a tool to provide both lung- and diaphragm-protective ventilation in humans.

SUPPLEMENTAL DATA

Online data supplement: https://doi.org/10.6084/m9.figshare.14077430

GRANTS

This work was supported by grants from the Royal Columbian Hospital Foundation, TB Vets, and MITACS. This work was supported by a grant from Lungpacer Medical Inc.

DISCLOSURES

Dr. Bassi is employed by Lungpacer Medical Inc. Dr. Reynolds is a minor investor in Lungpacer Medical Inc. and is listed on one of the original patents. All other authors have received salary support in order to execute this work which was funded in part from a grant from Lungpacer Medical Inc.

AUTHOR CONTRIBUTIONS

E.C.R. and S.C.R. conceived and designed research; E.C.R., T.G.B., K.C.F., M.O., M.N., J.C.W., and S.C.R. performed experiments; E.C.R., T.G.B., and J.C.W. analyzed data; E.C.R., T.G.B., K.C.F., and S.C.R. interpreted results of experiments; E.C.R. prepared figures; E.C.R. drafted manuscript; E.C.R. and S.C.R. edited and revised manuscript; E.C.R., T.G.B., K.C.F., M.O., M.N., J.C.W., and S.C.R. approved final version of manuscript.