Variable ventilation improves pulmonary function and reduces lung damage without increasing bacterial translocation in a rat model of experimental pneumonia

Thirty-two Wistar rats (weight, 300–410 g) were anesthetized under spontaneous breathing with 2% isoflurane (Isoforine^®; Cristália, Itapira, SP, Brazil) and randomly assigned to two groups: 1) Pneumonia (PA, n = 16), in which Pseudomonas aeruginosa 01 (ATCC27853, 5 × 10⁷ CFU diluted in 200 μL saline) was instilled intratracheally (i.t.) (see Additional file 1 for details of the development of the pneumonia model); and 2) Control, in which 200 μL saline was instilled i.t. (SAL, n = 16). Eight animals in the SAL and PA groups were not ventilated (NV) and served as controls for computation of lung damage score, ultrastructural damage score, and molecular biology analyses. After 24 h, animals were premedicated intraperitoneally (i.p.) with 10 mg/kg diazepam (Compaz, Cristália, Itapira, SP, Brazil), followed by 100 mg/kg ketamine (Ketamin-S+, Cristália, Itapira, SP, Brazil) and 2 mg/kg midazolam (Dormicum, União Química, São Paulo, SP, Brazil). An intravenous (i.v.) catheter (Jelco 24G, Becton, Dickinson and Company, New Jersey, NJ, USA) was inserted into the tail vein, and anesthesia induced and maintained with midazolam (2 mg/kg/h) and ketamine (50 mg/kg/h). Following local anesthesia with 2% lidocaine (0.4 ml), a midline neck incision and tracheostomy were performed. A second catheter (PE-50, Becton, Dickinson and Company) was then placed in the right internal carotid artery for blood sampling and gas analysis (Radiometer ABL80 FLEX, Copenhagen NV, Denmark), as well as monitoring of mean arterial pressure (MAP) (Networked Multiparameter Veterinary Monitor LifeWindow 6000 V; Digicare Animal Health, Boynton Beach, FL, USA). A 30-cm-long water-filled catheter (PE-205, Becton, Dickinson and Company) with side holes at the tip, connected to a differential pressure transducer (UT-PL-400, SCIREQ, Montreal, QC, Canada), was used to measure the esophageal pressure (Pes). The catheter was passed into the stomach and then slowly returned into the esophagus; its proper positioning was assessed with the “occlusion test” [14]. Animals were then paralyzed with 2 mg/kg pancuronium bromide i.v. (Cristália, Itapira, SP, Brazil), and lungs mechanically ventilated (Inspira, Harvard Apparatus, Holliston, MA, USA) in volume-controlled ventilation (VCV) mode with V_T = 6 mL/kg, respiratory rate = 80 breaths/min, FiO₂ = 0.4, and positive end-expiratory pressure (PEEP) = 5cmH₂O. Arterial blood gases and lung mechanics were analyzed (Baseline). SAL and PA groups were then randomly assigned to 2 h of conventional VCV or variable VCV (VV). Conventional ventilation settings were similar to those previously applied (V_T = 6 mL/kg, respiratory rate = 80 breaths/min, FiO₂ = 0.4, and PEEP = 5cmH₂O). At the end of the experiments (End), arterial blood gases were measured and 20 μL of peripheral blood was sampled for bacterial counts. Animals were killed by exsanguination through the arterial line, and their lungs extracted at PEEP = 5cmH₂O for light microscopy and molecular biology analyses.

Variable ventilation was applied as described in detail elsewhere [15, 16] Briefly, a sequence of randomly generated V_T values (normal distribution, mean = 6 mL/kg, coefficient of variation [CV] = 30%, n = 600) was applied in volume-controlled mode using a routine developed by our group (nVentInspira, Dresden, Germany). The sequence continuously looped itself until the end of the experiments. All other mechanical ventilator settings were kept unchanged: mean V_T = 6 mL/kg, respiratory rate = 80 breaths/min, FiO₂ = 0.4, and PEEP = 5cmH₂O. Figure 1 depicts representative tracings of airflow, volume, and airway pressure (Paw) during conventional ventilation (VCV, left column) and variable ventilation (VV, right column).

Airflow (\( \overset{.}{V} \)), as well as Paw and esophageal pressure (Pes) were continuously recorded throughout the experiments with a computer running customer-made software written in LabVIEW (National Instruments, Austin, TX) [17]. V_T was calculated by digital integration of \( \overset{.}{V} \). All signals were amplified in a four-channel signal conditioner (SC-24, SCIREQ, Montreal, QC, Canada). The mechanical properties of the lungs, namely elastance (E_L) and resistance (R_L), were calculated by fitting the signals to the equation of motion, according to transpulmonary pressure (P_L = Paw−Pes), as shown in Equation 1:where P₀,_L is P_L at end expiration.

The left lung was removed, fixed, and embedded in paraffin. Sections (4 μm thick) were cut and stained with hematoxylin and eosin. A lung damage score based on features commonly seen in pneumonia models was computed [18]. For this purpose, the following histological features were analyzed in the tissue sections: perivascular edema, septal neutrophils, and necrotizing vasculitis. Each feature was scored according to severity, with 0 denoting no effect and 4 denoting maximum severity, and extent, with 0 denoting no appearance and 4 denoting full involvement. The results were calculated as the product of severity and extent of each feature, ranging from 0 to 16, and added to yield the total lung damage score, ranging from 0 to 48.

Blood samples (20 μL) were seeded in Petri dishes with Tryptic Soy Agar growth medium (Fluka Analytical, St Louis, MO, USA). Manual counts of colony forming units (CFU) were performed after 24 h of incubation at 37 °C.

Quantitative real-time reverse transcription polymerase chain reaction (PCR) was performed to measure biomarkers associated with inflammation (interleukin [IL]-6 and cytokine-induced neutrophil chemoattractant [CINC-1]), type II alveolar cell mechanotransduction (surfactant protein-D [SP-D]), endothelial cell injury (angiopoietin [Ang]-2), and alveolar stretch (amphiregulin). The primers used are described in the online supplement (Additional file 1: Table S1). 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 RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s recommendations. RNA concentrations were measured by spectrophotometry in a Nanodrop ND-1000 system (ThermoScientific, Wilmington, DE, 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 an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, California, USA). Samples were run in triplicate. For each sample, the expression of each gene was normalized to the acidic ribosomal phosphoprotein P0 (36B4) housekeeping gene [19] and expressed as fold change relative to respective NV animals, using the 2^–∆∆ Ct method, where ΔCt = Ct_{reference gene} – Ct_target gene [20].

Sample size calculation was based on effect estimates obtained from previous studies in rodents using similar ventilator settings [8]. A sample size of eight animals per group would provide the appropriate power (1 − β = 0.8) to identify significant (α = 0.05) differences in respiratory system elastance between VCV and variable ventilation, taking into account an effect size d = 1.38, a two-sided test, and a sample size ratio = 1 (G*Power 3.1.9.2, University of Düsseldorf, Düsseldorf, Germany).

The Kolmogorov-Smirnov test with Lilliefors correction was used to assess the normality of data, whereas the Levene median test was used to evaluate the homogeneity of variances. For the pneumonia model, the Student t-test and Mann-Whitney U test were used for comparisons of parametric and nonparametric data respectively.

The present study expands the notion that VV is associated with beneficial effects on gas exchange and lung protection in respiratory failure. Since pneumonia is one of the major risk factors for ARDS development [2] and these patients frequently require mechanical ventilation, VV might represent a valuable strategy to improve pulmonary function and reduce lung damage without promoting further injury or bacterial translocation to the blood stream. Furthermore, in patients without lung injury, VV might be useful to prevent deterioration of lung function and increases in inflammatory markers, which could lead to further pulmonary complications. These issues warrant investigation in future experimental and clinical studies.

Some limitations of this study must be noted. First, pneumonia was induced by i.t. instillation of Pseudomonas aeruginosa, and our results cannot be extrapolated to other types of pulmonary infection. Nevertheless, in a 10-year retrospective study [21], 45.8% of patients had nosocomial-acquired pneumonia caused by Pseudomonas aeruginosa. Furthermore, lungs infected by other pathogens might also benefit from VV-induced responses, e.g., increased production of surfactant. Second, the data presented herein refer to the application of variable ventilation during controlled mechanical ventilation, not assisted ventilation, which might have yielded different results. Third, unlike in clinical settings, PEEP, respiratory rate, and FiO₂ were kept constant. However, as the main objective was to evaluate VV, confounding factors resulting from changes in ventilator settings were excluded. In this line, the level of PEEP used in the current study, while often used in rats, may not be directly extrapolated to the clinical setting. Nevertheless, it has been estimated that values of PEEP in rats should be multiplied by a factor of 2 to 2.5 [44], when comparing with humans. In our study, this corresponds to 10 to 12.5 cmH₂O, i.e., a moderate to high PEEP value in humans. Fourth, the observation time was relatively short (2 h of mechanical ventilation), precluding extrapolation of the findings to longer periods of ventilation. Finally, protein levels of biomarkers of VILI were not determined. Instead, we chose to assess expression of biomarker mRNA, because an experimental period of 2 h might not be sufficient to detect differences in protein levels [45‐48].