Background
A negative consequence of mechanical ventilation using lower tidal volumes (VT) in patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is alveolar collapse
Buchman
Therefore, in this study in pigs with oleic acid lung injury, we compared BVV to conventional CMV or CMV with a recruitment manoeuvre (CMV-RM) of 40 cm H2O of continuous positive airway pressure for 40 sec performed hourly for 5 hrs. This approach has been shown to improve oxygenation in patients with early ARDS who do not have any chest wall impairment
Methods
Experimental Preparation
Pigs (weighing 20–30 kg) received 0.6 mg atropine, 15 mg midazolam, and 300 mg ketamine intramuscularly for sedation. Isoflurane 5% in 100% oxygen was delivered via facemask to induce anaesthesia. When sufficient depth of anaesthesia was achieved, the pigs were intubated orotracheally with a 6.0 mm cuffed endotracheal tube. Mechanical ventilation was instituted with an Ohio 7000 ventilator (Ohio Medical, Madison WI) with minute ventilation adjusted to maintain a PaCO2 of 35–45 mm Hg. Anaesthesia was maintained with 2% isoflurane in 100% oxygen during surgical preparation. Intravenous rocuronium bromide (1 mg/kg/hr) was administered by continuous infusion for muscle relaxation. Lactated Ringer's solution was given intravenously during the surgical preparation and for the duration of the experiment.
A thermodilution pulmonary artery catheter (7.5-Fr) was inserted and advanced into the right external jugular vein until a satisfactory pulmonary capillary wedge tracing was obtained. Temperature was measured from the tip of the pulmonary artery catheter. The right femoral artery was cannulated for continuous pressure transduction and arterial blood gas (ABG) analysis. A 5-Fr single lumen femoral venous catheter was advanced into the inferior vena cava for infusion of oleic acid. A surgical tracheotomy was performed and the animal was switched to an Esprit® ventilator (Respironics Inc., Palo Alto CA) capable of delivering either CMV or BVV. The ventilator was set to deliver a square wave inspiratory flow pattern with an I:E ratio of 1:2. Isoflurane was discontinued, and a propofol/ketamine infusion at 10/2.5 mg/kg/hr substituted to maintain anaesthesia.
After a 30 cm H2O recruitment manoeuvre for 30 sec, animals were ventilated with a VT of 10 ml/kg at an FIO2 of 0.3, with 5 cm H2O of PEEP. Respiratory rate was adjusted to maintain PaCO2 between 35–45 mm Hg.
After 15 min to stabilize, baseline measurements were obtained. Haemodynamic measurements included mean arterial pressure (MAP), heart rate, central venous pressure (CVP), mean pulmonary artery pressure (MPAP), and pulmonary artery occlusion pressure (PAOP). All haemodynamic data were continuously recorded on a Gould 2600 Oscillograph (Gould, Cleveland, OH). Cardiac output (CO) was measured by thermodilution, in triplicate, at stated measurement periods.
A pneumotachograph (Model 3700; Hans Rudolph, Kansas City, MO) with the sensor immediately proximal to the tracheotomy was used to measure airway pressures and VT intermittently; this data was recorded using an advanced CODAS (Dataq Instruments, Akron, OH) data acquisition system.
Arterial and mixed venous gases were analyzed using a Radiometer ABL 500 (Copenhagen NV, Denmark). Arterial and mixed venous oxygen content, oxygen saturation, and haemoglobin concentrations were measured with a Radiometer OSM3 set for porcine blood.
Static respiratory system compliance (Crs) was measured in triplicate by clamping the expiratory limb of the ventilator circuit at the end of inspiration for 1 sec to obtain a plateau pressure. The VT used was that which the animal was receiving at that time.
Oleic Acid Lung Injury
Oleic acid was infused via the 5-Fr femoral venous catheter at 0.2 ml/kg/hr until PaO2 <60 mm Hg for two consecutive measurements (PaO2/FIO2 <200). Dopamine was started at 5 μg/kg/min and was titrated to keep the MAP >60 mmHg. When the oxygenation target was achieved, the oleic acid infusion was stopped and the PEEP was increased to 10 cm H2O. Ten minutes after the increase in PEEP, an arterial blood gas sample was obtained to determine if PaO2 had increased. PaO2 had to be >75 mm Hg but <90 mm Hg. This was considered to represent adequate lung injury, but indicate that collapsed alveoli could be recruited with the additional PEEP. If the PaO2 did not increase, the experiment was terminated. If the PaO2 increased to >90 mm Hg, additional oleic acid was infused until the PaO2 decreased to <60 mm Hg.
Ventilation Protocol
A low VT (7 ml/kg) protocol was initiated and the respiratory rate increased to 30 bpm. After 10–15 min arterial blood gas sampling was done to assess the stability of the PaO2. If the PaO2 remained stable, the animals were then randomized into one of three groups: conventional ventilation with a VT of 7 ml/kg (CMV); conventional ventilation with VT of 7 ml/kg with a 40 sec, 40 cm H2O recruitment manoeuvre performed hourly (CMV-RM). The recruitment manoeuvre was performed at end-expiration with the PEEP level maintained at 10 cm H2O at an FIO2 of 0.3; or biologically variable ventilation (BVV) with a mean VT of 7 ml/kg.
Following stable oleic acid lung injury, haemodynamic, gas exchange and respiratory system compliance (Crs) measurements were recorded and obtained hourly thereafter. Measurements in the CMV-RM group were obtained 5 min after each recruitment manoeuvre (RM). An additional measure of gas exchange and Crs was made in the CMV-RM group 50 min after the RM, to ascertain the duration of effect of the RM.
Wet:Dry Lung Weight Ratios
At the end of the experiment, a sternotomy was performed. Animals were sacrificed with a lethal dose of thiopental. The trachea was then clamped at end inspiration and the heart and lungs were removed
Surfactant Function Assays
Surfactant function was assessed on BAL fluid samples using a capillary surfactometer (Calmia Medical, Toronto, Canada) in the manner of Enhorning and colleagues
1. Raw BAL fluid surfactant function analysis – centrifuging at 200 g for 5 min to rid large debris, then the supernatant spun at 10,000 g and pellet resuspended in 100 μL saline and analyzed using the surfactometer.
2. Surfactant resuspended in 100 μL of saline after chloroform/methanol extraction. Chloroform/methanol extraction permits the lipid and phospholipid fraction to dissolve in the organic nonpolar solvent (chloroform) and the solvent evaporated to dryness. Volume of the final ratio for chloroform:methanol:water was 1:1:0.9. Following extraction, the pellet was resuspended and analyzed using the surfactometer.
The percentage of time that the capillary tube was open for 2 min was determined for each sample, in triplicate, then averaged. Standards were saline; 0% patent for a 2 min time period and bovine surfactant; >98% patent for a 2 min time period. Each sample was measured in a blinded fashion in both conditions.
Flow Cytometry Analysis
The BAL fluid sample was passed through a cell strainer and 100 μL was transferred into a 5 × 75 mm tissue culture tube. Red cells were lyses by the addition of 500 μL Optilyse C (Beckman Coulter, Mississauga, Canada) and following 15 min incubation at room temperature, the cell suspension was diluted by the addition of 500 μL Isoton II and 100 μL FlowCount Fluorospheres (Beckman Coulter). The cell suspension was immediately analyzed on a Beckman Coulter EPICS Altra cell sorter configured with a high-speed quartz flow cell tip and a water-cooled argon laser emitting 150 mW at 488 nm. Forward and side light scatter signals were employed to derive 2-parameter histograms which clearly defined the fluorescent beads and three populations of cells, subsequently defined as mononuclear cells, neutrophils and eosinophils. At least 20,000 cells were analyzed for each sample. The acquisition software provided with the instrument automatically calculated the concentrations of each cell population based on the number of events in each of the 4 analysis regions.
Cytokine Assays for IL-8 and -10 ELISA
The incubation times and washes were preformed as specified in each respective kit: (BioSource International, Camarillo, CA). Immunoassay kits for swine IL-8 and IL-10 were used. Sample incubation times were kept as constant as possible by pre-plating on a blank 96 well plate before transferring to the coated assay plate. ELISA plates were read at 450 nm by an SLT Rainbow plate reader (Lab Instruments, Research Triangle Park, NC). Standard curves and concentration calculations were performed according to kit directions.
Histological Assessment
For light microscopy, four blocks of tissue from the right lung – upper lobe, middle lobe, nondependent and dependent lower lobe, fixed in buffered formalin, processed and embedded in formalin. Sections were cut and stained with haematoxylin and eosin. Under light microscopy, a lung injury scoring system modified from Rotta et al.
Statistical Analysis
Parametric data were analyzed by repeated measures ANOVA using least squares means test matrices to identify differences within and between groups from group × time or group effects. Bonferroni's correction was applied where appropriate. Non-parametric comparison of the lung injury scores was by Kruskal-Wallis test. In all circumstances a p-value ≤0.05 corrected for multiple comparisons was considered significant.
Results
Twenty-five experiments were undertaken. One animal was discarded because oleic acid injury could not be established after 1.5 hrs of infusion. In the CMV-RM group, one animal died 2 hr into the experiment but data are included in analysis in this experiment up to time of the animal's demise. Complete experiments were done in twenty-three animals; (n = 8 for all three groups).
Temperature and Haemodynamic Data
Temperature and haemodynamic data are shown in Table 1: see
Table 1: Temperature and Haemodynamics
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Respiratory Gas and Derived Data
Significant differences were seen between groups for PaO2 over time (Figure
1a and b – Arterial Oxygenation and Respiratory System Compliance
1a and b – Arterial Oxygenation and Respiratory System Compliance. Arterial oxygen tension (PaO2) over time for the 3 groups (BVV in red; CMV in blue and CMV-RM in green) (a). The group × time interaction is p = 0.0001. No difference is seen between groups at baseline and following oleic acid infusion. By 2 hr the PaO2 is greater with BVV (+). There is no difference between CMV and CMV-RM. Respiratory system compliance over time for the 3 groups (b). The group × time interaction is p = 0.089. Least squares means tests revealed BVV had significantly greater compliance after 2 hr (+). There was no difference between CMV and CMV-RM.
Table 2: see
Table 2: Respiratory Gas and Derived Data
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Recruitment Effects on Oxygenation and Compliance
PaO2 tended to decrease immediately following a recruitment manoeuvre, but increased over time as measured at 50 min. PaO2 significantly decreased in RM4 and 5 (-11.1 ± 5.2 mm Hg and -5.2 ± 4.4 mm Hg respectively). Following recruitment, PaO2 increased slowly early in the experiment at RM2 and RM3 (10.1 ± 5.5 mm Hg and 11.8 ± 9.1 mm Hg respectively), but failed to do so in the later time periods. For respiratory system compliance, no statistically significant differences were seen at any time period over the course of the experiment in the CMV-RM group.
Airway Pressure Data
A group × time interaction for mean Paw was seen (p = 0.002) with Paw lowest with BVV (Paw at 5 hr was 14.3 ± 0.4 cm H2O with BVV; 15.1 ± 0.2 with CMV-RM and 15.1 ± 1.0 with CMV respectively). In all 3 groups, mean Paw increased following oleic acid. A more pronounced effect was seen with peak Paw (group × time; p = 0.0001) with mean peak pressures over time least with BVV (peak Paw at 5 hr was 23.2 ± 1.9 cm H2O with BVV; 28.8 ± 0.8 with CMV-RM and 28.6 ± 2.8 with CMV respectively). There was no group × time interaction for VT in ml/kg measured over time (p = 0.617).
Lung Histology
When the lung was scored in the upper, middle, nondependent and dependent lower lobes in the right lung, no differences were seen for any region by Kruskal-Wallis test between the 3 groups. In the dependent region of the lower lobes, injury was considerable with scores ranging between 9/28 and 22/28. Wet:dry weight ratios for the three groups were BVV; 8.5 ± 4.0, CMV; 8.9 ± 1.2, and CMV-RM; 11.5 ± 4.8; not significant between any group.
Capillary Surfactometry
Capillary surfactometry was done on raw BAL fluid and following chloroform/methanol extraction. There was no difference in surfactometry for raw BAL fluid between groups; the duration of capillary tube patency as an index of surfactant function was: 6.7 ± 14.3%, 0.7 ± 0.8% and 0.9 ± 1.7%; for BVV, CMV and CMV-RM respectively – not significant. Following chloroform/methanol extraction, surfactant function of the same fluid usually improved dramatically – see Figure
Capillary Surfactometry
Capillary Surfactometry. Box and whisker plots for capillary surfactometry results from both raw (blue) and chloroform/methanol extracted (red) BAL fluid and patency time (%) over 2 min – log scale. In most circumstances the raw BAL fluid has minimal surface activity – low capillary patency. The one animal with a high raw value was from the BVV group. This patency markedly improved with extraction suggesting reactivation of surfactant with removal of BAL oedema fluid, proteins, including cytokines and cellular debris. In 4 animals there was essentially no change in function with extraction. There were 2 animals in the CMV group, and one each in the BVV and CMV-RM in this population. No statistically different behaviour in surfactant function was seen between groups, either for raw or extracted surfactant function.
Flow Cytometry
The flow cytometry of BAL fluid indicate no significant difference between cell counts either in terms of neutrophils, monocytes or eosinophils between groups. Neutrophils were the predominant cell type, at 74% ± 15% of the totals. The total BAL cell counts were highly variable however, with neutrophil counts varying from a low of 187 to a high of 26,189 cells/μL – a 140-fold difference. There was no group effect for neutrophils; p = 0.741. The mean BAL neutrophil counts were 8237 ± 8738/μL for BVV, 12024 ± 12328/μL for CMV and 6492 ± 4219/μL for CMV-RM. BAL fluid volume was also measured based on gravity drainage from the lung over a 10 min period. Volume was not significantly different between groups and was 13.6 ± 16.8 mL with BVV, 15.1 ± 10.6 mL with CMV and 28.4 ± 23.2 mL with CMV-RM. The total neutrophil count in the BAL fluid was calculated as neutrophils/μL × BAL volume in μL for each animal. Mean total neutrophil counts were 1.38 × 108 for BVV, 1.82 × 108 for CMV and 2.34 × 108 for CMV-RM. There was no significant difference between groups.
ELISA Results and Cytometry Correlations
The ELISA results to assess the cytokine IL-8 concentration in BAL fluid indicated no statistically significant difference for the 3 groups: 564 ± 551 pg/mL with BVV; 653 ± 639 pg/mL with CMV and 300 ± 235 pg/mL with CMV-RM. As in the measurement of neutrophil counts, there was significant variability with IL-8 levels ranging over a 385-fold concentration difference. Such a broad range of data in both neutrophil counts and IL-8 concentration suggested the possibility of power law behaviour and correlation between these variables. A very strong power law relationship was found between IL-8 pg/mL and absolute neutrophil count/mL for pooled data; y = 77609x0.75; R2 = 0.85, n = 13. This power law relationship exceeded the linear (R2 = 0.60) and exponential (R2 = 0.41) curve fits.
The number of mononuclear cells from cell cytometry of BAL fluid was not different between groups: for BVV, 1346 ± 743 cells/μL; for CMV, 1767 ± 905 cells/μL; and for CMV-RM, 1068 ± 579 cells/μL. These cells presumably in large part represent alveolar macrophages. A power law relationship was seen between pooled data for a correlation between IL-8 concentration in pg/mL and monocyte count/mL – 1.0 × 10-6x1.38; n = 13, R2 = 0.83. In this situation the linear and exponential fits were R2 = 0.61 and R2 = 0.69 respectively. In this analysis, IL-8 concentration is presumed to be the dependent variable – being released by the monocytes (counts on the x-axis).
The IL-10 results from BAL fluid, as well, indicate no statistically significant difference for the 3 groups: 119 ± 173 pg/mL for BVV; 124 ± 146 pg/mL for CMV; and 149 ± 168 pg/mL for CMV-RM. Unlike IL-8 concentrations the range of variation for IL-10 concentrations was significantly less – the maximum range differing by only 16-fold. There was no power-law relationship found between IL-10 and absolute monocyte count for pooled data: y = 7.5x0.15; n = 13, R2 = 0.07.
Discussion
The main finding in this study of acute lung injury in a porcine model is that BVV significantly improved oxygenation and respiratory mechanics with no difference in indices of lung injury, inflammation or surfactant function compared to the more conventional ventilation techniques – CMV or CMV with a standard recruitment manoeuvre. The improvements seen with BVV were sustained over the course of the experiment; an effect not seen with recruitment. Such improvement was obtained at similar measured mean VT but at lower peak and mean airway pressure and greater respiratory system compliance.
These findings are similar to previously published results from our laboratory showing that BVV was superior to both CMV and CMV with sigh breaths. These sigh breaths were of no advantage in a model of healthy lung recruitment – lung reinflation after a controlled collapse for one hr with contralateral one-lung ventilation
Shunt fraction was significantly lower with BVV. Lynch et al.
A multimodal approach to assess lung injury, lung inflammation and surfactant function indicated no discernable differences between the 3 approaches to ventilation:
1. Light microscopy studies demonstrated no significant differences between groups for lung injury in any region examined – from upper to dependent lower lobes. This finding indicates no difference between groups in lung injury as assessed by histology, with the caveat that only small areas of lung in each region were examined in a condition known to be heterogeneous.
2. No difference in surfactant function was seen between groups as assessed by capillary surfactometry. Following oleic acid lung injury, surfactant function was markedly depressed when compared to a surfactant standard – see Figure
3. No difference between groups is seen for cell counts in the BAL fluid. There is considerable variation in this analysis and a full data set is not present. A significant number of studies of cell count could not be undertaken because of the nature of the BAL fluid. When thick with proteinaceous debris the samples often were unable to be prepared for meaningful cytometry analysis. Of the data represented, no difference in the proportion of neutrophils, monocytes or eosinophils is apparent for any of the 3 approaches to ventilation. Allen and Bates
4. IL-8 is considered to be the major neutrophil chemoattractant cytokine in lung diseases like ARDS
The above observations, collectively, indicate no fundamental differences between the ventilation strategies studied in regards oleic acid or superimposed ventilator associated lung injury. No one technique seems clearly advantageous. However, BVV alone improves oxygenation significantly following acute lung injury over time, an effect that was sustained, suggesting it is the best technique of the 3 studied to recruit atelectatic lung as assessed by greater PaO2 and respiratory system compliance. BVV has been shown to recruit in a pure model of lung collapse – re-expansion of lung following cessation of one lung ventilation
It could be argued that we have not chosen the optimal recruitment strategy to compare to the two other modes of mechanical ventilation. That said, we have chosen a well recognized approach to recruitment
The human variability file for respiratory rate used to program the ventilator is shown in Figure
Human Respiratory Rate Variability File
Human Respiratory Rate Variability File. Human breathing variability file used for the study. This is the raw data from a spontaneously breathing healthy female volunteer. The mean rate was 13.4 ± 2.0 breaths/min (shown as the red line). There are 1587 breaths in this file. With BVV, the ventilator is configured as a volume divider at a fixed minute ventilation so that respiratory rate × tidal volume product is constant. Thus the breath-by-breath volume related to instantaneous respiratory rate obtained from sequentially reading the above file in any given experiment is obtained from the minute ventilation/[(instantaneous breath rate/13.4) × chosen mean rate]. Analysis reveals that these data have fractal characteristics.
Conclusions
This study shows that BVV with a human variability file was superior to either CMV or CMV with a recruitment manoeuvre (CMV-RM) for sustained improvement in gas exchange and respiratory mechanics in a porcine model of acute lung injury. Prior work has demonstrated an advantage of BVV in a clinical setting of atelectasis
Authors' contributions
D.J.F. was responsible for conduct of the experiments as a fellow in the Anesthesia Laboratory. M.R.G. supervised conduct of the experiments, helped analyze data and helped write the paper. L.G.G. helped with the experiments, data retrieval and collation and table and figure production. J.A.T. did the histological assessments and analysis. B.M.M. supervised the cytokine assays and their interpretation and helped write the paper. E. K-Y. W. conducted the cytokine assays. C.H. did the surfactant assays while a fellow in the Oral Biology Laboratory. J.E.S. supervised and interpreted the results of the surfactant assays. W.A.C.M. conceived the study, analyzed and interpreted data and helped write the paper.