Background

Gradually changing respiratory rate (RR) during time to reduce ventilation-induced lung injury has not been investigated. The authors hypothesized that gradual, compared with abrupt, increments in RR would mitigate ventilation-induced lung injury and that recruitment maneuver before abruptly increasing RR may prevent injurious biologic impact.

Methods

Twenty-four hours after intratracheal administration of Escherichia coli lipopolysaccharide, 49 male Wistar rats were anesthetized and mechanically ventilated (tidal volume, 6 ml/kg; positive end-expiratory pressure, 3 cm H2O) with RR increase patterns as follows (n = 7 per group): (1) control 1, RR = 70 breaths/min for 2 h; (2) and (3) abrupt increases of RR for 1 and 2 h, respectively, both for 2 h; (4) shorter RR adaptation, gradually increasing RR (from 70 to 130 breaths/min during 30 min); (5) longer RR adaptation, more gradual increase in RR (from 70 to 130 breaths/min during 60 min), both for 2 h; (6) control 2, abrupt increase of RR maintained for 1 h; and (7) control 3, recruitment maneuver (continuous positive airway pressure, 30 cm H2O for 30 s) followed by control-2 protocol.

Results

At the end of 1 h of mechanical ventilation, cumulative diffuse alveolar damage scores were lower in shorter (11.0 [8.0 to 12.0]) and longer (13.0 [11.0 to 14.0]) RR adaptation groups than in animals with abrupt increase of RR for 1 h (25.0 [22.0 to 26.0], P = 0.035 and P = 0.048, respectively) and 2 h (35.0 [32.0 to 39.0], P = 0.003 and P = 0.040, respectively); mechanical power and lung heterogeneity were lower, and alveolar integrity was higher, in the longer RR adaptation group compared with abruptly adjusted groups; markers of lung inflammation (interleukin-6), epithelial (club cell secretory protein [CC-16]) and endothelial cell damage (vascular cell adhesion molecule 1 [VCAM-1]) were higher in both abrupt groups, but not in either RR adaptation group, compared with controls. Recruitment maneuver prevented the increase in VCAM-1 and CC-16 gene expressions in the abruptly increased RR groups.

Conclusions

In mild experimental acute respiratory distress syndrome in rats, gradually increasing RR, compared with abruptly doing so, can mitigate the development of ventilation-induced lung injury. In addition, recruitment maneuver prevented the injurious biologic impact of abrupt increases in RR.

Editor’s Perspective
• Several ventilatory parameters (tidal volume, peak and driving airway pressure, mechanical power) have been identified as key moderators of ventilator-induced lung injury

• The role of respiratory rate as an independent moderator is uncertain

• A rat model of mild acute lung injury, incorporating either a gradual or an abrupt increase in respiratory rate (with a preceding recruitment maneuver) greater than two different adaptation periods, evaluated postmortem histologic alveolar damage along with markers of lung inflammation, endothelial cell damage, and gene expression

• A gradual increase in respiratory rate resulted in evidence of less lung damage

• Biomarkers were higher with abrupt increases as well, although a preceding recruitment maneuver ameliorated this increase

Tidal volume (VT),1  respiratory system peak pressure,2  and respiratory system driving pressure 3,4  all have potential to injure the lungs of patients with the acute respiratory distress syndrome (ARDS). Respiratory rate (RR) is also an important ventilatory parameter to be controlled, although it is seldom integrated into ventilatory protocols in preclinical and clinical studies.5–7

It is widely known that, under some predisposing conditions characterized by mechanical heterogeneity, high RR can amplify microstresses and regional strains, perhaps causing ventilator-induced lung injury, as observed in experimental one-hit6–9  and two-hit10  models. At the cellular level, lung endothelial cells do not tolerate high-frequency conditions, showing changes in adhesion and releasing inflammatory cytokines.11  Whether gradually achieving a high RR might promote better strain distribution and consequently limit lung damage remains unknown. The biologic impact of abruptly increasing RR may depend on lung mechanical heterogeneity, for instance, simultaneous presence of fast and slow alveolar units. A recruitment maneuver theoretically may help protect against potentially injurious pulmonary effects of abruptly increasing RR. The aim of the current study in rats was to evaluate the biologic impact of gradually compared with abruptly increasing the rates of RR on lung damage in mild experimental ARDS. In addition, we hypothesized that a recruitment maneuver before abruptly increasing RR would attenuate any related injurious biologic impact.

This study was approved by the Ethics Committee of the Health Sciences Center, Federal University of Rio de Janeiro (CEUA: 015/19). All animals received care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research (Bethesda, Maryland) and the U.S. National Academy of Sciences (Bethesda, Maryland) Guide for the Care and Use of Laboratory Animals. The current study followed the Animal Research: Reporting of In Vivo Experiments guidelines for reporting of animal research.12  Animals were housed at a controlled temperature (23°C) and in a controlled light–dark cycle (12–12 h), with free access to water and food.

### Animal Preparation and Experimental Protocol

Fig. 1.

(A) Experimental design. (B) Timeline of experiments. (1) Control; RR = 70 breaths/min for 2 h; (2) abrupt increase of RR for 1 h; RR = 70 breaths/min during the first hour followed by an abrupt increase in RR to 130 breaths/min during the second hour; (3) abrupt increase of RR for 2 h; RR = 130 breaths/min during the first and second hour; (4) shorter RR adaptation; RR = 70 breaths/min during the first 30 min and gradual increase from 70 breaths/min up to 130 breaths/min (2 breaths/min increments) at 60 min, which remained during the second hour; (5) longer RR adaptation; RR = 70 breaths/min and immediately followed by a gradual increase from 70 breaths/min up to 130 breaths/min (1 breath/min increments) at 60 min, which remained during the second hour. Fio2, inspired fraction of oxygen; PEEP, positive end-expiratory pressure; RR, respiratory rate; VT, tidal volume.

Fig. 1.

(A) Experimental design. (B) Timeline of experiments. (1) Control; RR = 70 breaths/min for 2 h; (2) abrupt increase of RR for 1 h; RR = 70 breaths/min during the first hour followed by an abrupt increase in RR to 130 breaths/min during the second hour; (3) abrupt increase of RR for 2 h; RR = 130 breaths/min during the first and second hour; (4) shorter RR adaptation; RR = 70 breaths/min during the first 30 min and gradual increase from 70 breaths/min up to 130 breaths/min (2 breaths/min increments) at 60 min, which remained during the second hour; (5) longer RR adaptation; RR = 70 breaths/min and immediately followed by a gradual increase from 70 breaths/min up to 130 breaths/min (1 breath/min increments) at 60 min, which remained during the second hour. Fio2, inspired fraction of oxygen; PEEP, positive end-expiratory pressure; RR, respiratory rate; VT, tidal volume.

Close modal

In response to peer review, two additional control groups were included (n = 7/each): (1) control 2, abrupt increase of RR maintained for 1 h; and (2) control 3, in which the control-2 protocol was performed after recruitment maneuver (continuous positive airway pressure, 30 cm H2O for 30 s). We hypothesized that the application of the recruitment maneuver could open alveolar units otherwise collapsed at end-expiration, thereby reducing inhomogeneity among so-called slow and fast alveolar units.

### Data Acquisition and Processing

A pneumotachograph (internal diameter, 1.5 mm; length, 4.2 cm; distance between side ports, 2.1 cm) was connected to the tracheal cannula for airflow (V′) measurements.14  The pressure gradient across the pneumotachograph was determined using a SCIREQ differential pressure transducer (UT-PDP-02; SCIREQ, Montreal, Quebec, Canada). Airflow and airway pressures were recorded continuously throughout the experiments with a computer running custom-made software written in LabVIEW (National Instruments, USA).14  In brief, VT was calculated by digital integration of the airflow signal obtained from a custom-made pneumotachograph that was connected to the Y-piece of the ventilator tubing.

Both inspiratory and expiratory pauses for 5 s were done to accurately measure the respiratory system peak pressure, respiratory system plateau pressure, the respiratory system driving pressure, intrinsic PEEP, and extrinsic PEEP. The respiratory system plateau pressure was computed after a 5-s end-inspiratory pause according to the time point of evaluation. Extrinsic PEEP was measured at the pneumotachograph. Inspiratory airflow, RR, and VT were also measured. Mechanical power was also calculated by the following equation:

(1)

Cumulative power exposure was calculated in the 1-h and 2-h groups.1  All signals were amplified in a four-channel signal conditioner (SC-24, SCIREQ) and sampled at 200 Hz with a 12-bit analog-to-digital converter (National Instruments). Mechanical data were computed offline by a routine written in MATLAB (version R2007a; MathWorks, USA).1

### Lung Histology

#### Diffuse Alveolar Damage.

Lungs were extracted during an expiratory pause at PEEP, 3 cm H2O, and slices (4 μm thick) were cut and stained with hematoxylin and eosin. The diffuse alveolar damage score was quantified.15,16  Photomicrographs at magnifications of ×100, ×200, and ×400 were obtained from 10 nonoverlapping fields of view per section using a light microscope (Olympus BX51, Olympus Latin America, São Paulo, Brazil). Diffuse alveolar damage was quantified using a weighted scoring system by two investigators (A.C.F.F. and V.L.C.) blinded to group assignment and independently, as described elsewhere.17  The scores of each expert were combined to yield a final score by arithmetic averaging. In brief, scores of 0 to 4 were used to represent ductal overdistension, alveolar collapse, inflammation, and edema or hemorrhage, with 0 standing for no effect and 4 for maximum severity. In addition, the extent of each scored characteristic per field of view was determined on a scale of 0 to 4, with 0 denoting no visible evidence and 4 denoting widespread involvement. Scores were calculated as the product of the severity and extent of each feature on a range of 0 to 16. The cumulative diffuse alveolar damage score was calculated as the sum of each score characteristic and ranged from 0 to 64.1,16

#### Quantification of Heterogeneous Airspace Enlargement.

Airspace enlargement was assessed by measuring the mean linear intercept between alveolar walls at a magnification of ×400, as described elsewhere.18  To characterize the heterogeneity of airspace enlargement, the central moment of the mean linear intercept (D2 of mean linear intercept between alveolar walls) was computed from 20 airspace measurements19  according to equation (2)

$D2=μ⋅(1+σ2μ2+σ2)⋅(2+σ⋅γμ)$
(2)

where μ is the mean, σ is the variance of airspace diameters, and γ is the skewness of the diameter distribution. After D2 calculation, the heterogeneity index (β) was derived from D2 and mean linear intercept between alveolar wall values by their ratio.20  Quantification of heterogeneous airspace enlargement was performed by one expert investigator (A.C.F.F.) blinded to group assignment.

#### Immunohistochemistry.

To analyze the adherens junction protein E-cadherin, immunohistochemical procedures were performed on 4-μm-thick, paraffin-embedded lung sections using a mouse polyclonal antibody against E-cadherin (Catalog No. 610181; BD Transduction Laboratories, 1:250, USA). Visualization and image capture were performed using a light microscope (Eclipse E800, Nikon, Japan) coupled to a digital camera (Evolution, Media Cybernetics, USA), with the Q-capture 2.95.0 graphical interface (version 2.0 software. 5, Quantitative Imaging, Surrey, British Columbia, Canada). High-quality images (2048 × 1536 pixel buffer) were captured far from the airways and analyzed using ImagePro Plus software (version 4.5.1, Media Cybernetics). Quantification of immunohistochemistry was performed by two expert investigators (A.C.F.F. and L.A.) blinded to group assignment.

### Biologic Markers

Gene expression of biomarkers associated with inflammation (interleukin-6 [IL-6]),21  epithelial cell damage (club cell secretory protein [CC-16]),22  and endothelial cell damage (vascular cell adhesion molecule [VCAM-1])23  in lung tissue was measured by reverse transcriptase-polymerase chain reactions. Primer sequences are listed in Supplemental Digital Content 2, table 1 (https://links.lww.com/ALN/D9). Central slices of the right lung were cut, collected in cryotubes, flash-frozen by immersion in liquid nitrogen, and stored at −80°C. Total RNA was extracted from frozen tissues using the ReliaPrep RNA Tissue Miniprep System (Promega Corporation, USA) according to the manufacturer’s recommendations. RNA concentrations were measured by spectrophotometry in a Nanodrop ND-1000 system (Thermo Scientific, USA). First-strand cDNA was synthesized from total RNA using a Quantitec reverse transcription kit (QIAGEN, Hilden, Germany). Relative mRNA levels were measured with a SYBR green detection system in a Mastercycler ep realplex real-time polymerase chain reaction system (Eppendorf, Applied Biosystems, USA). Samples were run in triplicate. Expression of each gene was normalized to the housekeeping gene acidic ribosomal phosphoprotein P0 (36B4)24  and expressed as fold change relative to nonventilated animals, using the 2−∆∆Ct method, where ΔCt = Ct (target gene) − Ct (reference gene).25  Molecular biology data were analyzed by one investigator (M.M.) blinded to group assignment.

### Statistical Analysis

The number of animals was calculated according to a previous study.13  Taking into account that differences in diffuse alveolar damage score between low power or low VTversus high power or low VT achieved an effect size of d = 1.68, assuming a sample size ratio of 1 and a statistical power (1 − β = 0.8) to identify significant differences (α = 0.05), 7 animals per group were necessary. The sample size calculation was done in G*Power software (G* Power 3.1.9.2, University of Düsseldorf, Germany).

The primary outcome was the diffuse alveolar damage score, and secondary outcomes were heterogeneous airspace enlargement, immunohistochemistry, and molecular biology. To compare functional parameters over time, a fixed and mixed linear model based on random intercept was used for each animal, followed by Holm-Šidák comparison tests. For the linear mixed model, we used the terms group, time, and group × time interaction. To compare diffuse alveolar damage score, heterogeneous airspace enlargement, immunohistochemistry, and molecular biology, the Kruskal-Wallis test was performed, followed by Dunn’s comparison tests. Spearman correlation was done between cumulative mechanical power and cumulative diffuse alveolar damage score. Data that satisfied parametric assumptions were expressed as the mean and SD, and data that did not satisfy parametric assumptions as the median (interquartile range). Statistical significance was established at P < 0.05 (two-tailed). Statistical analysis was performed using GraphPad Prism for Windows (version 8.1.1, GraphPad, USA).

All animals survived to the end of the experiment (FINAL); thus, there were no missing data. Cumulative fluid administration was similar among groups (control, 11.4 ± 0.6 ml; abrupt increase of RR for 1 h, 11.7 ± 0.5 ml; abrupt increase of RR for 2 h, 11.8 ± 0.6 ml; shorter RR adaptation, 11.8 ± 0.9 ml, longer RR adaptation, 11.9 ± 0.7 ml). Oxygenation improved through time (Supplemental Digital Content 3, table 2, https://links.lww.com/ALN/D10). No differences in Paco2, arterial pH blood, or bicarbonate levels were observed among the groups. Mean arterial pressure remained higher than 90 mmHg, with no significant differences among groups throughout the experiments (Supplemental Digital Content 3, table 2, https://links.lww.com/ALN/D10).

Table 1.

Respiratory Parameters

Both abrupt increase of RR groups presented higher cumulative diffuse alveolar damage scores than the control group, due to increases in ductal overdistension, alveolar collapse, inflammation, and edema/hemorrhage (table 2, fig. 2). The cumulative diffuse alveolar damage score was reduced in the shorter and longer RR adaptation groups in comparison with both abrupt increases of RR groups. In addition, alveolar collapse and inflammation were lower in the shorter RR adaptation group than both abrupt increases of RR groups. Edema or hemorrhage was lower in the longer RR adaptation group than in both abrupt increases of RR groups (table 2, fig. 2). The diffuse alveolar damage score was lower in both adaptation groups (shorter and longer) compared with abrupt increase of RR for 1 h during 1 h (Supplemental Digital Content 5, table 3, https://links.lww.com/ALN/D12, Supplemental Digital Content 6, fig. 3, https://links.lww.com/ALN/D13). In addition, E-cadherin was fragmented, which denotes loss of epithelial integrity, in the abrupt increase of RR for 1 h during 1 h group (Supplemental Digital Content 7, fig. 4, https://links.lww.com/ALN/D14).

Table 2.

Diffuse Alveolar Damage Score and Alveolar Heterogeneity (β)

Fig. 2.

Histology of the lungs, visualized by hematoxylin-eosin staining at low, intermediate, and high magnification in control, abrupt increase of RR for 1 h, abrupt increase of RR for 2 h, shorter RR adaptation, and longer RR adaptation groups. Control animals exhibit preserved histoarchitecture of lung characterized by uniform distribution of the ventilation along ducts (A) and alveolar sacs (B), as well as perfusion along the alveolar capillaries (D), with sparse inflammatory cells (C). Lung tissue from the abrupt increase of RR for 1 h group exhibits prominent ductal overdistension (E) and alveolar collapse (F, black circles) with inflammation along the alveolar septa (G, black arrows) and minor degree of edema and alveolar hemorrhage (H, red arrows). Compared with the abrupt increase of RR for 1 h group, the abrupt increase of RR for 2 h was associated with a more inhomogeneous distribution of ventilation in ducts (I) and alveolar sacs (J), causing rupture of the alveolar septa in an emphysema-like pattern (J, black square), associated with more intense septal inflammation (K, black arrows), alveolar edema, and septal hemorrhage (L, red arrows). In contrast, the shorter and longer RR adaptation groups appear to exhibit a more protective effect of ventilation along the ducts and alveolar sacs, with minor ductal overdistension (M and Q, respectively) and alveolar collapse (N and R; black circles, respectively), with minor inflammation (O and S, black arrows, respectively) as well as perfusion along the alveolar capillaries (P and T; red arrows, respectively). RR, respiratory rate.

Fig. 2.

Histology of the lungs, visualized by hematoxylin-eosin staining at low, intermediate, and high magnification in control, abrupt increase of RR for 1 h, abrupt increase of RR for 2 h, shorter RR adaptation, and longer RR adaptation groups. Control animals exhibit preserved histoarchitecture of lung characterized by uniform distribution of the ventilation along ducts (A) and alveolar sacs (B), as well as perfusion along the alveolar capillaries (D), with sparse inflammatory cells (C). Lung tissue from the abrupt increase of RR for 1 h group exhibits prominent ductal overdistension (E) and alveolar collapse (F, black circles) with inflammation along the alveolar septa (G, black arrows) and minor degree of edema and alveolar hemorrhage (H, red arrows). Compared with the abrupt increase of RR for 1 h group, the abrupt increase of RR for 2 h was associated with a more inhomogeneous distribution of ventilation in ducts (I) and alveolar sacs (J), causing rupture of the alveolar septa in an emphysema-like pattern (J, black square), associated with more intense septal inflammation (K, black arrows), alveolar edema, and septal hemorrhage (L, red arrows). In contrast, the shorter and longer RR adaptation groups appear to exhibit a more protective effect of ventilation along the ducts and alveolar sacs, with minor ductal overdistension (M and Q, respectively) and alveolar collapse (N and R; black circles, respectively), with minor inflammation (O and S, black arrows, respectively) as well as perfusion along the alveolar capillaries (P and T; red arrows, respectively). RR, respiratory rate.

Close modal

The Supplemental Digital Content 8, fig. 5 (https://links.lww.com/ALN/D15), shows data on cumulative mechanical power in the control, abrupt increase of RR during 1 h, and longer and shorter adaptation groups. The cumulative power of abrupt increase of RR during 1 h was similar to that imparted to the control group ventilated for 2 h (Supplemental Digital Content 8, fig. 5, https://links.lww.com/ALN/D15 and Supplemental Digital Content 9, table 4, https://links.lww.com/ALN/D16). However, the diffuse alveolar damage score was higher in the abrupt increase of RR for 1 h group than in the control groups (Supplemental Digital Content 5, table 3, https://links.lww.com/ALN/D12). Furthermore, the cumulative power of longer and shorter RR adaptation groups ventilated for 2 h was higher than in the abrupt increase of RR for 1 h group. However, the diffuse alveolar damage score was lower in longer and shorter RR adaptation groups ventilated for 2 h than in animals exposed to abrupt increase of RR for 1 h.

The heterogeneity index (β) of airspace enlargement was higher in the abrupt increase of RR for 2 h group compared with the control group (P = 0.010) (table 2). The lower RR adaptation group showed lower β compared with both abrupt increase of RR groups (1 h and 2 h; P = 0.048 and P = 0.005, respectively) (table 2). Both abrupt increase of RR groups (1 h and 2 h) showed reduced alveolar integrity, as measured by lung tissue E-cadherin expression, compared with the control group (P = 0.026 and P = 0.017, respectively) (fig. 3). Similarly, the lower RR adaptation group showed better alveolar integrity compared with both abrupt increase of RR groups (1 h and 2 h; P = 0.041 and P = 0.026, respectively) (fig. 3).

Fig. 3.

Immunohistochemical staining for E-cadherin in lung tissue. Photomicrographs were taken at ×400 (AE). Scale bar, 100 μm. (F) Boxplots represent the median and interquartile range of 7 animals per group. Comparisons done by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (P < 0.05). *Significantly different from control. #Significantly different from abrupt increase of RR for 1 h. †Significantly different from abrupt increase of RR for 2 h. Control group did not differ from shorter (P = 0.999) and longer (P = 0.999) RR adaptation groups. Abruptly increasing RR for 1 h did not differ from abruptly increasing the RR for 2 h (P = 0.999). Abrupt increases of RR for 1 h did not differ from shorter RR adaptation (P = 0.267). Abruptly increasing RR for 2 h did not differ from shorter RR adaptation (P = 0.189). Shorter RR adaptation did not differ from longer RR adaptation (P = 0.999). RR, respiratory rate.

Fig. 3.

Immunohistochemical staining for E-cadherin in lung tissue. Photomicrographs were taken at ×400 (AE). Scale bar, 100 μm. (F) Boxplots represent the median and interquartile range of 7 animals per group. Comparisons done by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (P < 0.05). *Significantly different from control. #Significantly different from abrupt increase of RR for 1 h. †Significantly different from abrupt increase of RR for 2 h. Control group did not differ from shorter (P = 0.999) and longer (P = 0.999) RR adaptation groups. Abruptly increasing RR for 1 h did not differ from abruptly increasing the RR for 2 h (P = 0.999). Abrupt increases of RR for 1 h did not differ from shorter RR adaptation (P = 0.267). Abruptly increasing RR for 2 h did not differ from shorter RR adaptation (P = 0.189). Shorter RR adaptation did not differ from longer RR adaptation (P = 0.999). RR, respiratory rate.

Close modal

Abrupt increase of RR for 2 h was associated with a higher level of IL-6 (marker of inflammation) than in the control group (P = 0.038). IL-6 was reduced in the longer RR adaptation group compared with the abrupt increase of RR for 2 h group (P = 0.045). CC-16 (a marker of alveolar epithelial cell damage) and VCAM-1 (a marker of endothelial cell damage) were higher in both abrupt increases of RR groups (1 h [P = 0.025 and P = 0.017, respectively] and 2 h [P < 0.001 and P < 0.001, respectively]) compared with the control group. In addition, lung tissues from animals in the shorter and longer RR adaptation groups showed reduced VCAM-1 gene expression compared with the abrupt increase of RR for 2 h group (P = 0.013 and P = 0.045, respectively) (fig. 4).

Fig. 4.

Fig. 4.

Close modal

VCAM-1 gene expression was lower in animals subjected to recruitment maneuver before abrupt increase of RR (control 3) for 1 h than in the nonventilated group in and animals not subjected to recruitment maneuver before the abrupt increase in RR (control 2, P = 0.040 and P = 0.016, respectively). CC-16 gene expression increased in animals not subjected to recruitment maneuver before the abrupt increase in RR (P = 0.014), but not in those animals subjected to recruitment maneuver, compared with the nonventilated group (fig. 5).

Fig. 5.

Vascular cell adhesion molecule-1 (VCAM-1) and club cell secretory protein-16 (CC-16) gene expressions in the abrupt increase in RR from 70 to 130 breaths/min and ventilation for 1 h. One group was subjected to recruitment maneuver (continuous positive airway pressure: 30 cm H2O for 30 s), while the other was not. VCAM-1 gene expression was lower in those animals subjected to recruitment maneuver before the increment in RR from 70 to 130 breaths/min. CC-16 gene expression increased in those animals not subjected to recruitment maneuver, but not in animals subjected to previous recruitment maneuver. Comparisons were performed by Kruskal-Wallis test followed by Dunn’s multiple comparison test (P < 0.05). VCAM-1: Nonventilated group did not differ from abrupt increase of RR for 1 h without recruitment maneuver (P = 0.999); CC-16: Nonventilated group did not differ from abrupt increase of RR for 1 h with recruitment maneuver (P = 0.455), abrupt increase of RR for 1 h without recruitment maneuver did not differ from abrupt increase of RR for 1 h with recruitment maneuver (P = 0.552). RR, respiratory rate.

Fig. 5.

Vascular cell adhesion molecule-1 (VCAM-1) and club cell secretory protein-16 (CC-16) gene expressions in the abrupt increase in RR from 70 to 130 breaths/min and ventilation for 1 h. One group was subjected to recruitment maneuver (continuous positive airway pressure: 30 cm H2O for 30 s), while the other was not. VCAM-1 gene expression was lower in those animals subjected to recruitment maneuver before the increment in RR from 70 to 130 breaths/min. CC-16 gene expression increased in those animals not subjected to recruitment maneuver, but not in animals subjected to previous recruitment maneuver. Comparisons were performed by Kruskal-Wallis test followed by Dunn’s multiple comparison test (P < 0.05). VCAM-1: Nonventilated group did not differ from abrupt increase of RR for 1 h without recruitment maneuver (P = 0.999); CC-16: Nonventilated group did not differ from abrupt increase of RR for 1 h with recruitment maneuver (P = 0.455), abrupt increase of RR for 1 h without recruitment maneuver did not differ from abrupt increase of RR for 1 h with recruitment maneuver (P = 0.552). RR, respiratory rate.

Close modal

In mild experimental ARDS, we found that (1) the cumulative diffuse alveolar damage score decreased in both the shorter and longer adaptation groups compared with the abrupt increase of RR for 1 h and 2 h groups; (2) mechanical power was lower in the longer adaptation group than in the abrupt increase of RR for 1 h and 2 h groups; (3) the longer RR adaptation group showed less lung heterogeneity compared with the abrupt increase of RR for 1 h and 2 h groups; (4) alveolar integrity, measured by the amount of E-cadherin expression in lung tissue, was better preserved in the longer RR adaptation group compared with both abrupt increase of RR groups; (5) markers of lung inflammation (IL-6) and of epithelial and endothelial cell damage (CC-16 and VCAM-1) were higher in the abrupt increase of RR groups than in both RR adaptation strategies compared with the control group; (6) recruitment maneuver before the abrupt increase of RR prevented the rise in VCAM-1 and CC-16 gene expressions. Compared with an abrupt increase to a given RR, more gradual increments of RR to the same final level and time predictably decreases mechanical power, attenuating ventilator-induced lung injury, as measured by lung histology and molecular markers of damage. Cumulative power in the abrupt increase of RR for 1 h group was similar to that observed in the control 1 group, but diffuse alveolar damage score was higher in animals exposed to abrupt increase of RR for 1 h than in controls (Supplemental Digital Content 4, table 3, https://links.lww.com/ALN/D11). This result suggests an injurious effect of abruptly increasing RR for 1 h. Furthermore, the cumulative power of longer and shorter RR adaptation groups ventilated for 2 h was higher than in animals exposed to abrupt increase of RR for 1 h, but the diffuse alveolar damage score was lower in the former than in the latter, suggesting a protective effect of gradually increasing RR, even if more mechanical power accumulates over time; in other words, high levels of RR applied in an abrupt manner are associated with more lung-tissue injury.

We used a model of acute lung injury induced by intratracheal instillation of E. coli endotoxin,3,13,26,27  a well-established “first hit” for lung inflammation. After 24 h, different ventilatory RR strategies can superimpose a “second hit” to cause lung damage. The E. coli lipopolysaccharide model reproduces changes in lung function and histology comparable to human ARDS.28  Accordingly, the oxygenation index at BASELINE showed values consistent with mild ARDS (Supplemental Digital Content 2, table 2, https://links.lww.com/ALN/D9). In addition, the diffuse alveolar damage score, as well as markers of decreased epithelial integrity, were pronounced in the current study. Animal models have been used to advance the field of ventilator-induced lung injury pathophysiology because direct and invasive measurements cannot be performed in humans. For example, the pioneering study of Webb and Tierney29  on rats was revisited,30  and the results were confirmed. In preclinical and clinical studies, when VT and pressures are increased, higher respiratory rates predispose to ventilator-induced lung injury.7,8,10,31,32  However, to date, no study has compared whether gradual, compared with abrupt, increments in RR mitigate ventilator-induced lung injury in mild experimental ARDS.

We tested the hypothesis that applying an “adaptive” strategy that gradually approached a higher RR would produce less injury than an abrupt increase to that same RR; the latter is regularly applied in clinical settings.33  Our rationale was that RR represents an important component in the mechanical power formula, because it is not directly related to cumulative energy load over the span of multiple cycles.2,5,34  Although RR has received less attention, it has been associated experimentally with ventilator-induced lung injury.5,35  During protective ventilatory strategies in ARDS patients (e.g., VT ~6 ml/kg), it is usual practice to increase RR abruptly to control hypercapnia.33,36  However, higher levels of RR augment effective mechanical power, not only by increasing the number of cycles per unit time, but also by increasing microstresses and strains per cycle due to inadequate time to distribute parenchymal forces within the viscoelastic and mechanically heterogeneous injured lung.37  Dynamic compliance dropped for about 30 to 60 s after the abrupt RR increase. One likely explanation is that, during pressure-controlled ventilation, alveolar collapse in slow and/or poorly aerated alveolar units can increase strain heterogeneity and may cause lung injury.38,39  Alveolar collapse increases the tidal strain for the remaining fraction of the aerated lungs and acts together with short inspiratory time (due to high RR) to affect gas volume distribution.

An injured lung may be characterized by both slow and fast alveolar units. By promoting a gradual increase in RR, both alveolar units remain open and better accommodate stress (reduced airway pressures) for the same strain (VT). On the other hand, by promoting an abrupt increase in RR and shortening of inspiratory time, only fast alveolar units remain open, which may favor alveolar overdistension, heterogeneity, and lung damage. Thus, fast alveolar units that better accommodate strain tend to overdistend.2,7,34,40  After recruitment maneuver application, the fraction of slow alveolar units tends to decrease,41  as does the propensity of alveolar units to become atelectatic, which may decrease regional tidal strains and heterogeneity. Although the injurious biologic impact of abruptly increasing RR was observed in a heterogeneous lung, it was not detected when the lungs were previously subjected to recruitment maneuver. The injurious effects of abruptly increasing the RR may thus depend on the baseline condition of the lung, whether heterogeneous or homogeneous. In addition, the protective effect of the adaptation groups, whether shorter or longer, may rely on the gentle and continuous recruitment of slow alveolar units, while helping to prevent their becoming atelectatic. Although we have shown differences between shorter and longer VT adaptation in a previous study,1  no major differences were observed in shorter and longer RR adaptation groups. These differences may depend on the participation (and likely weights) of each variable in the mechanical power formula. For instance, it has been shown that increasing VT while keeping a stable and low mechanical power by proportionally reducing RR led to signs of ventilator-induced lung injury.42  This means that the potential of VT to cause lung damage was not fully annulled by RR modulation. We may emphasize that each variable has its own mechanism of injury and adaptations in heterogeneous ARDS lungs. However, because we did not perform experiments dealing with recruitment maneuver application followed by adaptation groups, this hypothesis should be tested in future preclinical studies.

### Possible Clinical Implications

Different components may contribute differently with mechanical power, such as respiratory system peak pressure, VT, respiratory system driving pressure, peak inspiratory flow, PEEP, and RR.1–4  During patient enrollment in the ARDS Network trial of VT (1996 to 1999),33  mechanical power was not a primary focus, and the potential contribution of an increase of RR to potential harm to the respiratory system was not considered. However, a recent observational retrospective study of 4,549 patients with ARDS showed that RR was associated with mortality,5  and should be considered when estimating the potential to inflict lung damage. Here, we expand this debate; not only is a given high RR injurious in mild experimental ARDS, but so, too, may be the rate of increase in RR toward that higher level. In addition, recruitment maneuver may play a relevant role in minimizing the injurious effects of abruptly increasing respiratory rate, by decreasing alveolar unit heterogeneity.

### Conclusion

In mild experimental ARDS in rats, we found that gradually increasing RR, compared with abruptly doing so, can mitigate the development of ventilator-induced lung injury. In addition, recruitment maneuver prevented the injurious biologic impact of abrupt increases in RR.

### Acknowledgments

The authors express their gratitude to the following people from the Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil: Andre Benedito da Silva, B.Sc., for animal care; Arlete Fernandes, B.Sc., for her help with microscopy; Camila Machado, Ph.D. student, for her help with microscopy; Maíra Rezende Lima, M.Sc., for her assistance with molecular biology analysis; and Moira Elizabeth Schöttler, M.Sc., Rio de Janeiro, Brazil, and Filippe Vasconcellos, M.Sc., São Paulo, Brazil, for editing assistance.

### Research Support

This study was supported by the Brazilian Council for Scientific and Technological Development (CNPq), the Rio de Janeiro State Research Foundation (FAPERJ E-26/202.766/2018, E-26/010.001488/2019), the São Paulo State Research Foundation (FAPESP 2018/20403-6), the Coordination for the Improvement of Higher Education Personnel (CAPES, 88881.371450/2019-01), and the Department of Science and Technology - Brazilian Ministry of Health (DECIT/MS).

### Competing Interests

The authors declare no competing interests.

Supplemental Digital Content 1, Figure 1. Experimental configuration, https://links.lww.com/ALN/D8

Supplemental Digital Content 2, Table 1. Oligonucleotide sequences of target gene primers, https://links.lww.com/ALN/D9

Supplemental Digital Content 3, Table 2. Arterial blood gases and mean arterial pressure, https://links.lww.com/ALN/D10

Supplemental Digital Content 4, Figure 2. Spearman correlation, https://links.lww.com/ALN/D11

Supplemental Digital Content 5, Table 3. Diffuse aveolar damage score, https://links.lww.com/ALN/D12

Supplemental Digital Content 6, Figure 3. Histology in abrupt increase of RR during 1 h, https://links.lww.com/ALN/D13

Supplemental Digital Content 7, Figure 4. E-cadherin in abrupt increase of RR during 1 h, https://links.lww.com/ALN/D14

Supplemental Digital Content 8, Figure 5. Cumulative power during ventilatory strategies, https://links.lww.com/ALN/D15

Supplemental Digital Content 9, Table 4. Respiratory parameters, https://links.lww.com/ALN/D16

Supplemental Digital Content 10, Figure 6. Cdyn,RS during abrupt increase in respiratory rate, https://links.lww.com/ALN/D17

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