Prone position prevents regional alveolar hyperinflation and mechanical stress and strain in mild experimental acute lung injury

Respiratory Physiology & Neurobiology 167 (2009) 181–188

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Prone position prevents regional alveolar hyperinflation and mechanical stress and strain in mild experimental acute lung injury Maria Cristina E. Santana a , Cristiane S.N.B. Garcia a , Débora G. Xisto a , Lilian K.S. Nagato b , Roberta M. Lassance c , Luiz Felipe M. Prota c , Felipe M. Ornellas c , Vera L. Capelozzi d , Marcelo M. Morales c , Walter A. Zin b , Paolo Pelosi e , Patricia R.M. Rocco a,∗ a

Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, RJ, Brazil Laboratory of Respiration Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, RJ, Brazil c Laboratory of Cellular and Molecular Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, RJ, Brazil d Department of Pathology, University of São Paulo, São Paulo, SP, Brazil e Department of Ambient, Health and Safety, University of Insubria, Varese, Italy b

a r t i c l e

i n f o

Article history: Accepted 6 April 2009 Keywords: Tissue stress and strain Ventilator-induced lung injury Lung mechanics Gas-exchange Lung histopathology

a b s t r a c t Prone position may delay the development of ventilator-induced lung injury (VILI), but the mechanisms require better elucidation. In experimental mild acute lung injury (ALI), arterial oxygen partial pressure (PaO2 ), lung mechanics and histology, inflammatory markers [interleukin (IL)-6 and IL-1␤], and type III procollagen (PCIII) mRNA expressions were analysed in supine and prone position. Wistar rats were randomly divided into two groups. In controls, saline was intraperitoneally injected while ALI was induced by paraquat. After 24-h, the animals were mechanically ventilated for 1-h in supine or prone positions. In ALI, prone position led to a better blood flow/tissue ratio both in ventral and dorsal regions and was associated with a more homogeneous distribution of alveolar aeration/tissue ratio reducing lung static elastance and viscoelastic pressure, and increasing end-expiratory lung volume and PaO2 . PCIII expression was higher in the ventral than dorsal region in supine position, with no regional changes in inflammatory markers. In conclusion, prone position may protect the lungs against VILI, thus reducing pulmonary stress and strain. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The prone position has been proposed to improve gas-exchange and respiratory mechanics in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) patients, although recent prospective randomized clinical trials (Gattinoni et al., 2001b; Guerin et al., 2004; Mancebo et al., 2006; Fernandez et al., 2008) have failed to demonstrate an improvement in clinical outcome. Experimental studies on healthy (Broccard et al., 2000; Nakos et al., 2006) and lung injured animals (Broccard et al., 1997) ventilated with high tidal volume and positive end-expiratory pressure (PEEP) have shown that prone position causes less extensive histological changes in dorsal regions when compared to supine position. In contrast, other studies using high tidal volume ventilation have

∗ Corresponding author at: Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute of Federal University of Rio de Janeiro, Centro de Ciências da Saúde, Avenida Carlos Chagas Filho, 373, Bloco G-014, Ilha do Fundão – 21941-902, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 6530; fax: +55 21 2280 8193. E-mail address: [email protected] (P.R.M. Rocco). 1569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2009.04.006

demonstrated a homogeneous distribution of lung injury independent of position, suggesting that prone position is effective in delaying ventilator-induced lung injury (VILI) (Nishimura et al., 2000; Valenza et al., 2005). The beneficial effects of prone position leading to the reduction of VILI are related to many factors: (a) a more homogeneous distribution of transpulmonary pressure gradient due to changes in the lung–thorax interactions and direct transmission of the weight of the abdominal contents and heart (Albert et al., 1987; Mutoh et al., 1992) yielding a redistribution of ventilation; (b) increased end-expiratory lung volume resulting in a reduction in stress and strain (Gattinoni et al., 2003; Valenza et al., 2005); and (c) changes in regional perfusion (Lamm et al., 1994; Richter et al., 2005). So far, however, no study has correlated the changes in regional lung distribution of blood flow and aeration with the biochemical response induced by modifications in lung parenchyma stress and strain. Therefore, we undertook this experimental study to compare the effects of prone and supine positions on the distribution of aeration and blood flow, lung mechanics, end-expiratory lung volume, oxygenation, and spatial distribution of lung injury.

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2. Methods 2.1. Animal preparation A total of 100 female Wistar rats [220 ± 10 (S.E.M.) g] were used. Animals were randomly assigned to the control (C) or acute lung injury (ALI) groups where sterile saline (0.9% NaCl, 5 ml/kg BW) or paraquat (25 mg/kg BW, suspended in saline solution with total volume equal to 0.5 mL) were intraperitoneally injected (i.p.). After 24 h, animals were sedated (diazepam 5 mg, i.p.), anesthetized (thiopental sodium 20 mg/kg BW, i.p.), tracheotomised, paralysed (pancuronium 2 mg/kg, i.v.), and mechanically ventilated (Samay VR15, Universidad de la Republica, Montevideo, Uruguay) with the following parameters: tidal volume (VT) = 5 mL/kg, airflow = 6 mL/s, respiratory frequency = 100 breaths/min, inspiratory to expiratory ratio = 1:2, fraction of inspired oxygen (FiO2 ) = 0.21, and zero positive end-expiratory pressure (ZEEP). A polyethylene catheter (PE-10) was introduced into the femoral artery for blood sampling. Blood (300 ␮L) was drawn into a heparinized syringe for arterial oxygen partial pressure (PaO2 ) analysis (AVL Biomedical Instruments, Roswell, GA, USA). Lung mechanics were then obtained (Baseline) and animals were randomized to either supine or prone positioning. Lung mechanics and histology were studied in 28 rats (n = 7/group). After 1-h ventilation period, PaO2 and lung mechanics were analysed. At the end of the experiments, lungs were prepared for histology and interleukin (IL)-6, IL-1␤, and PCIII mRNA expressions in lung tissue were measured. This study was approved by the Ethics Committee of the Carlos Chagas Filho Institute of Biophysics, Health Sciences Center, Federal University of Rio de Janeiro. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences, USA. 2.2. Respiratory mechanics Airflow, and tracheal (Ptr) and oesophageal (Poes) pressures were measured. Tidal volume was calculated by digital integration of flow signal (Riva et al., 2008). Transpulmonary pressure (PL) was calculated as Ptr–Poes. Lung resistive (P1, L) and viscoelastic/inhomogeneous (P2, L) pressures, and static elastance (Est, L) were computed by the end-inflation occlusion method (Bates et al., 1988). Pulmonary mechanics measurements were performed 10 times in each animal, and analysed using ANADAT data analysis software (RHT-InfoData). 2.3. Lung histology Immediately after the determination of lung mechanics in supine or prone position, a laparotomy was done, and heparine (1000 IU) was intravenously injected. The trachea was clamped at end-expiration in ZEEP in supine or prone position. The abdominal aorta and vena cava were sectioned, yielding a massive haemorrhage and quick death. The right lung was tied off at ZEEP immersed in 3% buffered formaldehyde and the left lung cut into strips and frozen in liquid nitrogen. After fixation, the tissue blocks were cut horizontally into 1-cm thick slices in ventral–dorsal order and embedded in paraffin. From each segment, 4-␮m thick slices were then cut and underwent haematoxylin–eosin staining. Morphometric analysis was performed with an integrating eyepiece with a coherent system consisting of a grid with 100 points and 50 lines (known length) coupled to a conventional light microscope (Axioplan, Zeiss, Oberkochen, Germany). Hyperinflated lung regions were defined as

alveolar ducts, sacs or alveoli with an internal cross-section diameter greater than 120 ␮m (Silva et al., 1998). Areas of collapse were defined as alveoli with rough or plicate walls. The volume fraction of the lung occupied by hyperinflated or collapsed alveoli or normal pulmonary areas (those not presenting overdistended or plicate walls) were determined by the point-counting technique (Gundersen et al., 1988; Weibel, 1990; Rocco et al., 2001; Riva et al., 2008) made across 10 random non-coincident microscopic fields at a magnification of 200×. Two investigators, who were unaware of the origin of the material, examined the samples microscopically. The slides were coded and examined only at the end of all measurements. In four other experimental groups (n = 7/group), India ink (1.0 mL) was injected into the pulmonary artery at a pressure of 25 cm H2 O. Black ink particles were identified in blood vessels. Immediately after the removal of the right lungs, these were also cut horizontally into 1-cm thick slices in ventral–dorsal order. In each slice, four 1 cm3 blocks, two from the apex and two from the base, were chosen at random and excised. From each block, 4-␮m slices were then cut and stained with haematoxylin–eosin. We analysed ventral and dorsal sections of apex and base of right lung parenchyma. For each segment, the ratios between alveolar aeration and lung tissue (alveolar aeration/tissue ratio) and between the India ink staining and lung tissue (India ink/tissue ratio) were computed. Briefly, points falling on alveolar space or India ink were counted and divided by the points falling on lung tissue in each microscopic field at a magnification of 200× (20 random non-coincident microscopic fields). Thus, the distribution of air and blood flow was quantified. We also measured surface-to-volume ratio to validate alveolar aeration/tissue ratio measurement. Lung surface density can be estimated by intersection counting, so that the probability of a test line of given length to intercept the alveolar septum is directly proportional to its surface density in the lung parenchyma. The surface (S)-to-volume (V) ratio was, therefore, calculated by S/V = 2I/L, where I is the number of intersections of the alveolar septum with a test line and L is the cumulative length of the test line obtained by L = Pd/d, where P is the number of points counted over the airspaces and d is the length of the test line in ␮m. The surface-to-volume (S/V) ratio is usually considered an estimate of shape of gas-exchanging parenchyma (Silva et al., 1998). 2.4. Expression of IL-6, IL-1ˇ and PCIII mRNA Lung parenchyma strips (3 mm × 3 mm × 10 mm) were cut from the left lungs and quick-frozen by immersion in liquid nitrogen. Quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR) was performed to measure the relative levels of expression of lung inflammatory cytokines and type III procollagen genes. Total RNA was extracted from the left lung frozen lung tissue, using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. The RNA concentration was measured by spectrophotometry. First-strand cDNA was synthesized from total RNA using a M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA) as instructed by the manufacturer. PCR primers for target gene were purchased from Invitrogen. Relative mRNA levels were measured with a SYBR green detection system on an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA). All samples were measured in triplicate. We measured the expression levels of type III procollagen, interleukin-6 (IL-6) and IL-1␤. The relative amount of expression of each gene was calculated as a ratio compared with the reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

M.C.E. Santana et al. / Respiratory Physiology & Neurobiology 167 (2009) 181–188 Table 1 Arterial oxygen partial pressure. PaO2 (mmHg)

Supine

Prone

Baseline End

91 (87–96) 86 (82–91)

89 (86–94) 99 (97–100)a , b

ALIc Baseline End

73 (60–79) 71 (60–78)

70 (61–76)a 83 (77–90)a , b

183

assessed by Two-way ANOVA followed by Tukey’s test considering C and ALI and different position as the two factors for analysis. Nonparametric data were analysed using Two-way ANOVA on ranks followed by Dunn’s post hoc test. A p value < 0.05 was considered significant.

C

3. Results In ALI, PaO2 was lower compared to controls (Table 1). After 1-h ventilation, in both controls and ALI, PaO2 did not change in supine position but increased in prone position. ALI significantly increased Est, L, P1, L, and P2, L compared to controls (Table 2). In controls, after 1-h ventilation, lung mechanics, except for P2, L, deteriorated independent of positioning. However, in ALI, all mechanical parameters were higher after 1h ventilation in supine position. In prone position, Ptot, L, P1, L, and P2, L were also higher while Est, L remained stable. Prone position resulted in lower Est, L, Ptot, L, and P2, L compared to supine position (Table 2). End-expiratory lung volume (EELV) was lower in ALI compared to C, both in supine (0.57 ± 0.04 mL and 1.15 ± 0.14 mL, respectively) and prone (0.76 ± 0.07 mL and 1.06 ± 0.02 mL, respectively) (p < 0.05). However, prone position led to higher EELV compared to supine only in ALI (p < 0.04). The fraction area of alveolar collapse was higher in ALI groups compared to controls. In supine position, the amount of atelectasis was greater in dorsal region (43% vs. 32%, p < 0.05) while hyperinflated areas were observed in ventral region (10%). Prone position decreased the amount of atelectasis both in ventral and dorsal regions (Table 3, Fig. 1). Alveolar aeration/tissue ratio was lower in ALI than controls. Additionally, in ALI alveolar aeration/tissue ratio was higher in supine than in prone position in ventral regions, both in apex and base. In contrast, in dorsal regions alveolar aeration/tissue ratio was lower in supine than in prone position in the base (Fig. 1). India ink/tissue ratio was higher in prone than supine position both in ventral and dorsal regions from apex to base (Figs. 2 and 3), suggesting that there were more particles in a given tissue volume in the prone position than in the supine position.

Arterial oxygen partial pressure (PaO2 ) values [median (range)] in control (C) and acute lung injury (ALI) groups. Data were measured immediately before (baseline) and after 1 h mechanical ventilation (end) both in supine (S) and prone (P) position. a Prone versus supine (p < 0.05). b End versus baseline (p < 0.05). c All PaO2 data from ALI group were significantly different from those of the control (p < 0.05).

2.5. End-expiratory lung volume Four additional experimental groups (n = 7/group) were submitted to the aforementioned protocols and end-expiratory lung volume (EELV) analysed. For this purpose, a laparotomy was done, the trachea was clamped at ZEEP in supine or prone position, and abdominal aorta and vena cava were sectioned. Lungs were removed en bloc, and weighed. A saline displacement technique was used to measure EELV (Scherle, 1970). Briefly, a jar containing saline solution and a submerged weight was placed on a laboratory scale, which was then adjusted to zero. Lungs were tied both to a laboratory stand and the weight by a thread and completely submerged in the saline solution. The liquid displaced by the submerged lungs directly expressed the weight gain registered on the scale, since the specific gravity of saline differs no more than 2–3% of the lung density (Scherle, 1970). EELV was computed subtracting the lung weight by the liquid displaced. 2.6. Statistical analysis SigmaStat 3.0 statistical software package (Jandel Corporation, San Raphael, CA, USA) was used. Differences among the groups were Table 2 Respiratory mechanical parameters. Variables

Baseline

End

Supine

Prone

Supine

Prone

C Ppeak, rs cmH2 O Pplat, rs cmH2 O Ppeak, L cmH2 O Pplat, L cmH2 O Est, L cmH2 O/mL Ptot, L cmH2 O P1, L cmH2 O P2, L cmH2 O

6.67 4.54 4.42 3.14 3.17 1.28 0.58 0.70

± ± ± ± ± ± ± ±

0.18 0.16 0.15 0.13 0.10 0.15 0.08 0.10

7.05 4.89 4.86 3.51 3.48 1.35 0.59 0.76

± ± ± ± ± ± ± ±

0.17 0.13 0.15 0.15 0.16 0.10 0.03 0.08

7.75 5.23 5.43 3.90 3.82 1.53 0.76 0.77

± ± ± ± ± ± ± ±

0.28# 0.25# 0.18# 0.22# 0.23# 0.17# 0.12# 0.07

7.90 5.59 5.52 3.90 3.90 1.62 0.83 0.78

± ± ± ± ± ± ± ±

0.23# 0.18# 0.25# 0.28# 0.26# 0.08# 0.13# 0.09

ALI* Ppeak, rs cmH2 O Pplat, rs cmH2 O Ppeak, L cmH2 O Pplat, L cmH2 O Est, L cmH2 O/mL Ptot, L cmH2 O P1, L cmH2 O P2, L cmH2 O

10.66 7.53 8.20 5.82 5.71 2.38 1.00 1.37

± ± ± ± ± ± ± ±

0.28 0.23 0.33 0.50 0.51 0.09 0.09 0.10

9.81 6.84 7.46 5.36 5.25 2.10 0.99 1.12

± ± ± ± ± ± ± ±

0.31 0.35 0.32 0.36 0.33 0.12 0.08 0.06

14.01 9.81 11.66 8.20 8.12 3.46 1.30 2.16

± ± ± ± ± ± ± ±

0.29# 0.28# 0.12# 0.92# 0.95# 0.18# 0.07# 0.13#

10.93 7.26 8.57 5.76 5.82 2.81 1.37 1.45

± ± ± ± ± ± ± ±

0.29# 0.27# 0.15# , ** 0.31** 0.38** 0.13# , ** 0.14# 0.09# , **

Values are means (±S.E.M.) of 7 rats in control (C) and acute lung injury (ALI) groups. Data were measured immediately before (baseline) and after 1 h mechanical ventilation (end) both in supine (S) and prone (P) position. rs: respiratory system. L: lung. Ppeak: peak pressure. Pplat: plateau pressure. Est: static elastance. Ptot, P1, P2: total, resistive, and viscoelastic pressures, respectively. * All ALI data were significantly different from control (p < 0.05). ** Prone versus supine (p < 0.05). # End versus baseline (p < 0.05).

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Table 3 Morphometrical parameters. Variables

ALI*

C Supine

Normal (%) Collapse (%) Hyperinflation (%)

Prone

Ventral

Dorsal

Ventral

98.2 ± 0.4 1.8 ± 0.5 0±0

97.3 ± 0.8 2.7 ± 0.6 0±0

96.3 ± 3.5 3.6 ± 0.9 0±0

Prone**

Supine Dorsal 97.5 ± 4.5 2.5 ± 0.6 0±0

Ventral

Dorsal

Ventral

Dorsal

68.7 ± 3.6 32.2 ± 3.8 10.1 ± 3.6

57.2 ± 0.8# 42.8 ± 9.7# 0 ± 0#

75 ± 9.2 25 ± 12.3 0±0

78.2 ± 9.5 21.8 ± 7.6 0±0

The volume fraction of the lung occupied by normal or collapsed or hyperinflated structures in control (C) and acute lung injury (ALI) groups. All values were computed in 10 random, non-coincident fields per rat. Values are mean (±S.E.M.) of 7 animals in each group. * All lung morphometrical data from ALI group were significantly different from those of the control (p < 0.05). ** All lung morphometrical data in prone position were significantly different from those of supine in ALI group (p < 0.05). # Dorsal versus ventral (p < 0.05).

ALI animals presented significantly smaller S/V ratios (p < 0.001) independent of supine or prone position, indicating that the amount of gas-exchanging surface available for a given lung volume is reduced. Additionally, S/V ratio is greater in ventral than dorsal regions in supine position with no significant changes in prone position. This finding is coherent with the presence of hyperinflation in ventral region (Fig. 4). In ALI, PCIII mRNA expression was higher compared to controls in supine and prone positions, both in ventral and dorsal lung regions. In controls, positioning did not affect PCIII mRNA expression, but, in ALI, supine position led to higher PCIII mRNA expression in the ventral compared to dorsal regions (Fig. 5). Prone position resulted in no significant differences in PCIII mRNA expression between ventral and dorsal lung regions. Additionally, IL-6 and IL-1␤ were higher in all regions in ALI than controls, but no regional

Fig. 1. Alveolar aeration/tissue ratio in ventral and dorsal segments of apex and base lung parenchyma, in control (C) and acute lung injury (ALI) groups after 1 h mechanical ventilation both in supine (S) and prone (P) position. Boxes show interquartile (25–75%) range, whiskers encompass range, and horizontal lines represent median values. *All ALI data were significantly different from control (p < 0.05). ␨ Base versus apex (p < 0.05).

differences in expression were found between supine and prone position (data not shown). 4. Discussion Our study demonstrates that, in the present experimental mild ALI model, prone position: (a) improved oxygenation, lung mechanics, and end-expiratory lung volume; (b) increased the amount of normal alveoli, while decreasing atelectasis in both ventral and dorsal regions, (c) reduced the amount of hyperinflation in the ventral region; (d) yielded more homogenous distribution of blood flow between ventral and dorsal lung regions; and (e) diminished the activation of PCIII mRNA expression in the ventral region. In the present study, ALI was induced by paraquat, a herbicide that accumulates predominantly in the lung and induces alveolar epithelial damage due to its action on type II pneumocytes.

Fig. 2. India ink staining/tissue ratio in ventral and dorsal segments of apex and base lung parenchyma in control (C) and acute lung injury (ALI) groups after 1 h mechanical ventilation both in supine (S) and prone (P) position. Boxes show interquartile (25–75%) range, whiskers encompass range, and horizontal lines represent median values. *All ALI data were significantly different from control (p < 0.05).

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Fig. 3. Photomicrographs of ventral and dorsal segments of apex and base lung parenchyma stained with haematoxylin–eosin in control (C) and acute lung injury (ALI) groups after 1 h mechanical ventilation both in supine (S) and prone (P) position. Arrows indicate the presence of India ink staining. Original magnification: 100×.

This model leads to a well reproducible lung injury characterized by alveolar collapse, interstitial oedema, and hyaline membranes, without alveolar oedema (Rocco et al., 2001), and presents an amount of atelectasis similar to that observed in human ALI/ARDS (Gattinoni et al., 2006). Previous experimental studies have investigated the mechanisms of the beneficial effects of prone position on gas-exchange as well as the protection from VILI from high tidal volumes in both healthy (Nishimura et al., 2000; Broccard et al., 2000; Valenza et al., 2005; Nakos et al., 2006) and injured (Broccard et al., 1997) lungs. In contrast, our study analyses both healthy and ALI animals ventilated with low tidal volume and ZEEP to minimize possible interactions between mechanical ventilation and prone positioning. The application of PEEP may lead to regional lung strain and stress influencing the effects induced by positioning (Gattinoni et al., 2003; Richard et al., 2008), although we cannot rule out that the use of ZEEP in our study may conversely promote atelectasis, particularly in dependent lung regions. This could have provided a larger signal differential between prone and supine groups. Additionally, animals were ventilated with air to prevent reabsorption atelectasis (Rothen et al., 1995) and reduce possible hyperoxia-induced lung injury (Kulkarni et al., 2007). In ALI group, prone position led to an increase in end-expiratory lung volume. The effects of prone position on EELV are controversial (Lee et al., 2005; Valenza et al., 2005) and variations in findings may be attributed to differences between measurement methods, animal species, ALI models, and ventilatory settings. The increase in EELV was accompanied by a significant reduction of the amount of atelectasis in both dorsal and ventral regions

in the ALI group (Table 3). The ratio between alveolar aeration and lung tissue (alveolar aeration/tissue ratio) was further evaluated (Fig. 1) along the vertical gradient from ventral to dorsal regions and from apex to base. Most previous studies have expressed the volume of alveolar aeration, using a point-counting technique, as volume density (VV ) of aerated alveolar spaces, using total parenchyma as reference volume (Zhou et al., 2000). Other studies have quantified alveolar aeration using other techniques (Mercer et al., 1987; Silva et al., 1998). In the present study, we counted the number of points falling on alveolar space and divided by the number of points falling on lung tissue in each microscopic field. This method used to quantify alveolar aeration has not been previously performed although similar results were obtained using other validated techniques such as surface-to-volume ratio (Silva et al., 1998) (Fig. 4). In the ventral regions alveolar aeration/tissue ratio was lower in prone than in supine position in both apex and base. However, in dorsal regions, alveolar aeration/tissue ratio was higher in prone compared to supine in the base, with no significant changes in the apex. Therefore, in prone position, a lower alveolar aeration/tissue gradient between ventral and dorsal regions was observed, suggesting a more homogeneous distribution of transpulmonary pressure. In supine position, even with lower tidal volume ventilation, ventral regions presented not only higher alveolar aeration/tissue ratio but also lung hyperinflation. Nevertheless, in prone position no hyperinflation was present. The different behaviour between ventral and dorsal regions at apex and base may be related to the distribution of the intrapleural pressure gradient, as well as lung and chest wall shape interactions (Liu et al., 1990; Osmon, 1995). Although we did not directly measure regional transpulmonary pressure, it has been

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Fig. 4. Surface-to-volume ratio [S/V (%)] in control (C) and acute lung injury (ALI) groups after 1 h mechanical ventilation both in supine (S) and prone (P) position. All values were computed in 10 random, non-coincident fields per rat. Values are mean (±S.E.M.) of 5 animals in each group. *All ALI data were significantly different from control (p < 0.05).

reported (Valenza et al., 2005) that during prone position, the lung is significantly wider and somewhat shorter compared to supine position, suggesting that the gradient of pleural pressure is different and more homogeneous in prone animals. Furthermore, it is interesting to note that the rat showed more significant modifications in dorso-ventral gradients of aeration with change of position than

Fig. 5. Real-time polymerase chain reaction analysis of type III procollagen mRNA (PCIII), mRNA expression of rat lung tissue in ventral and dorsal segment of lung parenchyma in control (C) and acute lung injury (ALI) rats during prone and supine position. Animals from each group were ventilated for 1 hour in supine (S) or prone (P) position. Data are normalized to GAPDH expression. The y-axis represents fold increase compared with C [non-ventilated (NV) animals]. Values are means (±S.E.M.) (n = 4). *All ALI data were significantly different from control (p < 0.05). **Prone versus supine (p < 0.05).

expected, despite its height of only 3–4 cm. It has been described that the redistribution of aeration is markedly dependent on the height of the lung from ventral to dorsal, due to a greater redistribution of regional transpulmonary and superimposed pressure on the alveoli (Gattinoni et al., 1991). However, our data suggest that position may play a relevant role independent of lung height. Some of these results are in line with previous experimental studies using different images techniques (Albert et al., 1987; Musch and Venegas, 2005; Valenza et al., 2005; Glenny et al., 2007). However, none of them have reported hyperinflation related to position in ALI, probably due to the inability of these techniques to detect hyperinflation (Gattinoni et al., 2001a). Thus, using lung morphometry, we were able to quantify not only the amount of alveolar collapse in different lung regions but also evaluate the ratio between alveolar aeration and lung tissue, and quantify hyperinflation. Regional distribution of blood flow was analysed through India ink technique (Butler and Kleinerman, 1970); however, this technique has never been used to quantify regional distribution of blood flow morphometrically. We found that, in control and ALI groups, regional distribution of blood flow was higher in prone compared to supine position independent of the region (ventral or dorsal). The increase in India ink/tissue ratio in prone position may be explained by: (1) constant India ink distribution and less volume of tissue; (2) increased India ink distribution with constant volume of tissue; and (3) increased India ink distribution and decreased volume of tissue. In the present study, we observe both an increase in India ink distribution and a reduction in the volume of tissue, leading to an increase in India ink/tissue ratio. In contrast, previous studies have reported that regional distribution of blood flow is predominantly in dorsal areas (Guerin, 2006; Musch et al., 2007; Richard et al., 2008). These differences could be also attributed to the size of the animal chest wall minimizing the impact of gravity on lung blood flow distribution and reducing the hypoxic vasoconstriction effects on lung vasculature, which could increase the distribution of blood flow both in ventral and dorsal regions (Richard et al., 2008). These pulmonary morphometrical changes resulted in an improvement in lung mechanics in prone position. In this line, prone position avoided the increase of lung static elastance after 1 h mechanical ventilation. Although viscoelastic/inhomogeneous pressure increased with time course in prone position, it was lower than in supine (Table 2). Prone position may have reduced the regional stress by leading to an improvement in lung static elastance and hence diminishing inspiratory transpulmonary pressure for the same inflated tidal volume. Prone position may have also reduced regional strain due to: (1) increased end-expiratory lung volume, (2) higher regional alveolar/tissue ratio in both ventral and dorsal lung regions and (3) reduced alveolar/tissue gradient from ventral to dorsal regions, which would yield a more homogeneous distribution of alveolar inflation (i.e. reduced the ratio between the regional inspiratory tidal volume and the end-expiratory volume). Since stress and strain have been proposed as important mechanisms determining VILI, we hypothesized that prone position could lead to a reduction in regional injury, even at low tidal volume ventilation in ALI (Gattinoni et al., 2003; Valenza et al., 2005). Two mechanisms are thought to contribute to the development of VILI during mechanical ventilation: alveolar hyperinflation in the non-dependent lung regions and shear stress and strain imposed by opening and stretching of collapsed lung in the dependent regions (Slutsky, 1999). Recent experimental reports have demonstrated that prone position may be effective in reducing (Broccard et al., 1997, 2000; Nakos et al., 2006) or delaying (Nishimura et al., 2000; Valenza et al., 2005) VILI. Some authors (Broccard et al., 2000; Nakos et al., 2006) have shown that the dorsal regions are more subject to VILI in supine position due to alveolar collapse leading to

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shear stress while, during prone position, a less severe but homogeneous injury has been observed. On the other hand, Nishimura and colleagues have found a homogeneous distribution of lung injury independent of the position (Nishimura et al., 2000). To better understand the role of lung hyperinflation and alveolar collapse in different lung regions, IL-6, IL-1␤, and PCIII mRNA expressions were measured in ventral and dorsal regions. PCIII mRNA expression is an early marker of lung parenchyma remodelling (Farias et al., 2005; Garcia et al., 2004; de Carvalho et al., 2007; Riva et al., 2008) and is higher in lungs submitted to elevated airway pressures (Parker et al., 1997; Farias et al., 2005; de Carvalho et al., 2007; Riva et al., 2008), high inflation (Berg et al., 1997) or cyclic mechanical strain (Garcia et al., 2004). In our study, supine position resulted in high PCIII mRNA expression in ventral regions. These changes may be associated with lung hyperinflation causing tensile stress and strain. However, these degrees of stress and strain were not high enough to induce changes in IL-6 or IL-1␤. Interestingly, the shear stress induced by alveolar collapse in dorsal region did not stimulate IL-6, IL-1␤ or PCIII mRNA expressions, even though no PEEP was used. Similar to our study, De Carvalho and colleagues have described, in a model of ALI induced by oleic acid in rats, that PCIII expression is higher in non-dependent regions that exhibited overdistension, while these changes are partially attenuated by prone positioning. Additionally, there is no significant regional difference in the expression of IL-1␤ (de Carvalho et al., 2007). Tsuchida et al. (2006) have also demonstrated that alveolar injury is maximal in the non-dependent region of non-injured ventilated lungs, consistent with redistribution of ventilation from atelectatic to non-atelectatic areas resulting in overinflation injury. During prone position such regional morphological and molecular changes in ventral and dorsal regions were no longer observed. Our study has several limitations which must be taken into account. In the present study, a specific experimental model of mild ALI was induced by paraquat. Thus, we do not know if similar results can be obtained in other experimental models of ALI, with different degrees of lung injury or in larger animals. Additionally, the duration of the experiment is short, just 1 h, which hinders assessment of possible long-term effects of prone position. Furthermore, the ventilation was set in the absence of PEEP, which could have maximized the effects of the prone position. Therefore, we cannot exclude different results by using different PEEP levels. Few studies have investigated regional distribution of blood flow in rats by changing positioning. We used India ink to analyse lung distribution of blood flow morphometrically, although this is an “unconventional”, non-validated technique. The delivery of India ink to the vasculature could depend on cardiac output, pulmonary vascular resistance, and the timing of the injection. Furthermore, post-mortem India ink analysis of distribution could be influenced by tissue preparation, cutting, and mounting. However, similar behaviour was observed both in control and ALI groups, suggesting that this approach may be considered valid. Although other imaging-based modalities such as magnetic resonance microscopy, microcomputed tomography, micro positron emission tomography, and microSPECT using real-time perfusion measurements have been used to investigate regional lung distribution of blood flow, they have not been validated in rats. In conclusion, in the experimental model of mild ALI and ventilatory settings utilized in the present study, prone position led to increased end-expiratory lung volume, less atelectasis, more homogeneous distribution of aeration and increased distribution of blood flow in both ventral and dorsal regions, resulting in improvement in lung mechanics and oxygenation compared to supine position. During low tidal volume ventilation, prone position reduced alveolar hyperinflation and PCIIII mRNA expression in ventral regions. Therefore, our data suggest that prone position may lead to reduced

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