Effects of prone and supine position on oxygenation and inflammatory mediator in a hydrochloric acid-induced lung dysfunction in rats

Purpose: To compare the effectiveness of mechanical ventilation of supine versus prone position in hydrochloric acid (HCl)-induced lung dysfunction. Methods: Twenty, adult, male, Wistar-EPM-1 rats were anesthetized and randomly grouped (n=5 animals per group) as follows: CS-MV (mechanical ventilation in supine position); CP-MV (mechanical ventilation in prone position); bilateral instillation of HCl and mechanical ventilation in supine position (HCl+S); and bilateral instillation of HCl and mechanical ventilation in prone position (HCl+P). All groups were ventilated for 180 minutes. The blood partial pressures of oxygen and carbon dioxide were measured in the time points 0 (zero; 10 minutes before lung injury for stabilization), and at the end of times acid injury, 60, 120 and 180 minutes of mechanical ventilation. At the end of experiment the animals were euthanized, and bronchoalveolar lavages (BALs) were taken to determine the contents of total proteins, inflammatory mediators, and lungs wet-to-dry ratios. Results: In the HCl+P group the partial pressure of oxygen increased when compared with HCl+S (128.0±2.9 mmHg and 111.0±6.7 mmHg, respectively) within 60 minutes. TNF-α levels in BAL do not differ significantly in the HCl+P group (516.0±5.9 pg/mL), and the HCl+S (513.0±10.6 pg/mL). Conclusion: The use of prone position improved oxygenation, but did not reduce TNF-α in BAL upon lung dysfunction induced by HCl.


Introduction
Gastric acid aspiration is a common etiology of acute lung injury (ALI) and is initially characterized by a chemical "burn" of the pulmonary epithelium, with a subsequent influx of extracellular fluid into the alveolar space that leads to pulmonary edema.Hence, acid aspiration-content is an important cause of acute respiratory distress syndrome (ARDS) 1 .
Acute lung injury (ALI) and ARDS are serious complications in trauma and surgical patients 2,3 .In spite of improvements in critical care and ventilator management, mortality rates have been reported to remain as high as 35% to 40% 4,5 .
A variety of ventilatory maneuvers are employed to combat it, such as mechanical ventilation with prone and/or supine position, positive end expiratory pressure (PEEP), alveolar recruitment, high-frequency ventilation and non-ventilatory maneuvers such as the administration of nitric oxide and surfactant, fluids management, corticoids [6][7][8] .
The use of prone ventilation has been shown to improve aeration and decrease alveolar shunt in an animal model 9 .Many clinical and experimental studies have since been carried out, but the mechanism responsible for the improvement remains highly controversial 4,10 .
During anesthesia and paralysis, the prone position determines a more homogeneous distribution of the gravitational gradient for alveolar inflation 11 .
Following aspiration other symptoms can be observed, with microvascular lung injury, hypoxemia, increase alveolar protein content and inflammatory response, etc.
The purpose of this study was to test the hypothesis whether mechanical ventilation with prone position improves arterial oxygenation and decreases local inflammatory dysfunction on an experimental model of ALI.

Surgical procedure
All procedures were approved by the Institutional Research Committee at Federal University of São Paulo, in accordance with the National Institutes of Health (NIH) Guidelines Regarding Animal Experimentation and the principles of the Brazilian College on Animal Experimentation.
After anesthesia, a tracheotomy was performed and a 14G cannula was inserted approximately 1 cm above the carina.A hind paw vein (used for drug infusions and compensation of fluid losses) and the left internal carotid artery (used to obtain arterial blood for gases determination) were exposed and cannulated using a PE-50 tubing (Intramedic Polyethylene Tubing, Becton Dickinson, NJ, USA).Normothermia was maintained with an underlying electric hotplate (37 ± 0.6oC [mean±SD]) 1 .

Mechanical ventilation (MV)
After reaching a surgical plane of anesthesia and muscle relaxation, the animals were ventilated using a rodent ventilator (683 model, Harvard Apparatus; MA, USA) for 3 h using a tidal volume (V T ) set at approximately 6 mL/kg, a positive end-expiratory pressure (PEEP) of 5 cmH 2 O, a fraction of inspired oxygen (FiO 2 ) of 1.0, a respiratory rate (RR) of 30-50 cycles per minutes and an inspiration/expiration (I/E) ratio of 1:1.A venous catheter was placed in the jugular vein to allow the infusion of isotonic saline solution and sodium pentobarbital whenever needed 1 .
The animals were divided into 4 different groups (n=5 in every group).The first group (CS-MV) received only conventional MV in supine position; the second group (CP-MV) received only conventional MV in prone position; the third group (HCl+S) received bilateral instillation of HCl and MV and in supine position; the fourth group (HCl+P) received HCl instillation and MV in the prone position for 180 minutes.The animals were maintained in the supine and/or prone position for 10 minutes, hereby defined as the baseline phase.In order to be included in the remainder of this study, each rat had to exhibit an initial arterial partial pressure of oxygen [PaO 2 ] > 300 mmHg.

Acid instillation lung injury
After 10 min connected in the ventilator for stabilization, the lung injury was performed using a 0.1 N HCl solution (2 mL/kg) instilled in the endotracheal tube in the supine position with the rat disconnected from the ventilator, the aspiration of HCl solution was performed in an inverted Trendelenburg position.When PaO 2 < 150 mmHg the protocol was started 1 .

Measurements Arterial blood gases measurement
Arterial blood gases (PaO 2 and arterial partial pressure of carbon dioxide [PaCO 2 ]) were determined in blood samples collected with sterile vented plastic syringes (PICO 70, Radiometer, Copenhagen, Denmark) and measured at the times acid injury, 60, 120 and 180 min using an automatic AVL-Compact3 device (Roche Diagnostic, Mannheim, Germany).

Wet/dry (W/D) weight ratio of lungs
After bronchoalveolar lavage, the right lung was placed in a drying oven for 72 h at 60°C and then re-weighed.The W/D ratios of lungs were then calculated.

Bronchoalveolar lavage (BAL)
At the end of 3 h, the animals were euthanized (1 mL/100 g bw T-61 Euthanasia Solution, Hoechst & Roussel).A sternotomy was performed, and the lungs and appending structures were removed and weighed.The left lung was tied, and the right lung was washed three times with 20 mL/kg cold sterile phosphate-buffered saline (PBS; pH 7.2-7.4).Lavage fluids were kept separate and centrifuged (at 400 x g for 10 min) at 4ºC, and the supernatants were frozen immediately on dry ice and stored at -80ºC 1 .

Protein leakage
An 1-mL aliquot of BAL was used to measure the total protein content using specific kits (QUANT-iT Protein Assay, Invitrogen, CA, USA) and a standard equipment for readings (QUBIT Fluorometer, Invitrogen, CA, USA).

Statistical analysis
Data were expressed as means ± standard deviation (SD).Values were compared between groups using an ANOVA test for multiple comparisons, and repeated-measures ANOVA was used to compare measurements among groups along the different time periods.Statistical analyses were performed using a standard computer software package (GraphPad Prisma, GraphPad Software, San Diego, CA, USA).P-values less than 0.05 were considered statistically significant.

Results
None of the animals died before the end of the experiment.The groups did not differ significantly in terms of the variables studied during the baseline period, either in terms of gas exchange, nor did the body weights differ at the end of experiment.
Between control groups (CS-MV and CP-MV) no significant differences were found concerning the studied variables.Differences could be observed after induced lung injury in groups HCl+S and HCl+P when compared with their respective controls (Table 1, Figures 1, 2 and 3).

Blood gases analyses
After 60 min of mechanical ventilation the PaO 2 values were higher in the HCl+P group compared with HCl+S group (128.0±2.9 versus 110.0±6.7 mmHg, respectively).In the time point 120 min the PaO 2 were higher in the HCl+P (152.0±2.5 mmHg) compared with HCl+S (146.0±4.2 mmHg) (Table 1), and the difference was observed at the time 180 min when comparing HCl+S versus HCl+P (P<.001;Table 1).At the times 60, 120 and 180 minutes the PaO 2 differ when compared HCl+S and HCl+P with respectively control group (Table 1).PaCO 2 values were lower in the HCl+P group compared to the HCl+S group (Table 1) in the times 60, 120 and 180 min.No significant differences were observed among HCl groups at the acid injury time point.

W/D weight of lungs, total proteins, and TNF-α in the BAL
W/D ratios were higher in the HCl+S group as compared to the HCl+P group (P<.05; Figure 1).The total proteins in BAL were lower in the HCl+P group compared to HCl+S group [38.0±2.9 mg/mL versus 47±2.3 mg/mL, respectively; P<.001].In the CS-MV group and in CP-MV, the total protein values in BAL were 26.0±1.6 mg/mL and 23.0±2.3mg/mL, respectively (Figure 2).The TNF-α concentration in BAL was significantly higher in the group HCl+S and HCl+P when compared with the respective control group (P<.001), but did not differ between HCl-injured groups (Figure 3).

Discussion
The present study confirm that prone position promoted the recovery of PaO 2 after HCl instillation.Decreased levels of total proteins in BAL accompained this alleviating effect on lung injury.Since West's accurate descriptions of the physiology of respiration and the ventilation/perfusion zones 12 , position change has been advocated to promote redistribution of gas, recruit alveoli, and redistribute pulmonary blood flow 13 .
Prone position was initially introduced in healthy anesthetized and paralyzed subjects for surgical specific reasons.Since then, it has been used during acute respiratory failure to improve gas exchange.The interest on prone position during ALI/ARDS progressively increased, even if the mechanisms leading to a respiratory improvement are not yet completely understood.Our results demonstrated the ameliorating effect on the recovery gas exchange in the group with lung injury and ventilated in the prone position.
Reports of prone positioning to improve oxygenation in patients with ALI/ARDS date back nearly 30 years 14 .Animal studies have demonstrated improved oxygenation and ventilation perfusion ratios, along with restoring aeration, decreasing shunt, and preserving perfusion 1,9,15 .In addition, prone positioning has been shown to eliminate compression of the lungs by the heart 16 .
TNF-α is an early-response, pro-inflammatory cytokine that has been shown to be integral in the pathogenesis of other lung injury models that are neutrophil-mediated [17][18][19][20] .TNF-α leads to activation of leukocytes and induces the expression of endothelial adhesion molecules that play a major role in the leukocyte-endothelial interactions that lead to neutrophil migration into the alveoli 21 .Our results demonstrated a small reduction in the TNF-α levels in the group HCl+P, but these effects did not differ significantly in comparison with the HCL+S group.
Computerized tomography of the chest shows that, while ARDS injuries are heterogeneous, they primarily affect the dorsal regions of the lungs 22 .Therefore, in the supine position, pulmonary ventilation without PEEP is primarily distributed to the ventral region of the lungs.The ratio of ventilation between ventral and dorsal regions is around 2.5:1.With the addition of PEEP, the distribution of ventilation becomes progressively more homogeneous.It means that ratio of close to 1:1 with PEEP at 20 cmH 2 O 23 .

Conclusion
The use of the prone position improves arterial oxygenation and reduces the lung edema in a rat model of acid injury.
No differences were observed in the time Injury in the PaO 2 and PaCO 2 levels among all groups.*P<0.05 compared with HCl+S group in the PaO 2 and PaCO 2 measurements (time 60 min); #P<0.05 compared with HCl+S group in the PaO 2 measurements (time 120 min); §P<0.05 compared with HCl+S group in the PaO 2 and PaCO 2 measurements (time 180 min).

FIGURE 1 -
FIGURE 1 -Effect of position on lungs wet to dry ratios from rats, mechanically-ventilated controls (C-MV) and instilled with HCl.*P<.05 versus the HCl+S group.Values are presented as mean ± SD.Values are presented as mean ± SD

FIGURE 2 -
FIGURE 2 -Effect of position on total protein levels in BAL from rats, mechanically-ventilated controls (C-MV) and instilled with HCl.*P<0.05 versus the HCl+S group.Values are presented as mean ± SD

FIGURE 3 -
FIGURE 3 -Effect of position on TNF-α levels in BAL from rats, mechanically-ventilated controls (C-MV) and instilled with HCl.*P<0.05 versus the CS-MV group, and #P<.05 versus the CP-MV group.Values are presented as mean ± SD

TABLE 1 -
Effects of PEEP after hydrochloric acid (HCl) on the arterial blood levels of PaO 2 and PaCO 2 in the time points Injury, 60, 120 and 180 minutes.Data are shown as mean±SD