Experimental Lung Research, Early Online, 1–13, 2015 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2014.1003436

ORIGINAL ARTICLE

Regional lung tissue changes with mechanical ventilation and fluid load Cristiana Marcozzi,1 Andrea Moriondo,1 Eleonora Solari,1 Marcella Reguzzoni,1 Paolo Severgnini,2 Marina Protasoni,1 Alberto Passi,1 Paolo Pelosi,3 and Daniela Negrini1 1

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Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy Department of Surgical Sciences and Integrated Diagnostics, IRCCS San Martino IST, University of Genoa, Genoa, Italy A B STRA CT Purpose: To investigate the regional gravity-dependent impact of mechanical ventilation and fluid overload on lung extracellular matrix (ECM) in healthy lungs. Materials and Methods: The glycosaminoglycans (GAGs) composition of the ventral and dorsal lung parenchyma was determined in anesthetized supine healthy rats mechanically ventilated for 4 hours in air: (a) at low (∼7.5 mL/kg) or high (∼ 23 mL /kg) tidal volume (VT ) and 0 cmH2 O positive end-expiratory pressure (PEEP); (b) at low or high VT at 5 cmH2 O PEEP and (c) with or without 7 mL /(kg·h) intravenous saline infusion. Results: Mechanical ventilation degraded lung ECM, with alveolar septa thinning and structural GAGs disorganization. Low VT ventilation was associated with significant tissue structure changes in both ventral and dorsal lung regions, while high VT mainly affected the dependent ones. PEEP decreased ECM injury mainly in the ventral lung regions, although it did not prevent matrix fragmentation and washout at high VT . Intravascular fluid load increased lung damage prevalently in the ventral lung regions. Conclusion: Mechanical ventilation and fluid load may cause additive injuries in healthy lungs, mainly in ventral regions. KEYWORDS hydrodynamic tissue stress, lung extracellular matrix, mechanical tissue stress, pulmonary interstitium, regional lung injury

INTRODUCTION The pulmonary extracellular matrix (ECM) is a mesh of fibrous macromolecules immersed in the interstitial extravascular fluid. Fibrillar macromolecules like collagen type I and III or elastin essentially play structural functions, providing tensile strength and/or lung elastic recoil. The so called “non fibrillar” macromolecules, including proteoglycans (PGs) and hyaluronic acid are also very important in the matrix, as they: (a) provide flexible bridges between fibrillar macromolecules; (b) fill intermolecular spaces, stabilizing the matrix scaffold and providing resistance to both external tissue compression and interstitial fluid expansion; (c) interact with macromolecules of the Received 1 September 2015; accepted 27 December 2015 Address correspondence to Daniela Negrini, Ph.D., University of Insubria, Department of Surgical and Morphological Sciences, Via J.H. Dunant 5, Varese 21100, Italy. E-mail: [email protected]

vascular basement membrane, limiting fluid transport across the endothelium and providing molecular sieving. Hence, disturbance of the PG homeostasis may lead to development of pulmonary edema and, eventually, lung injury. PGs include families of multi-domain core proteins covalently bond to glycosaminoglycan (GAG) chains, whose polyanionic nature accounts for the physical PGs properties. In the lung, PGs are represented mainly by: (a) large PGs (versican) containing chondroitin sulfate or dermatan sulphate GAG chains (CS/DS-GAGs) with marked hydrophilic properties, which play an important role in cell–matrix interaction, matrix synthesis, and wound healing [1, 2] and (b) smaller PGs (perlecan) containing heparan sulfate GAGs (HS/HEGAGs), typically arranged in the vascular basement membranes where they interact with collagen IV, providing a perivascular selective sieve, which minimizes the endothelial permeability to fluid and solutes [1, 3]. In addition, HS-PGs may bind glycoproteins 1

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involved in cell–matrix adhesion, growth factors, enzymes and their inhibitors, viral coat proteins and regulatory nuclear proteins [2], thus controlling cell migration, proliferation, and gene expression, wound healing and angiogenesis [1]. The ECM also seems to play an important role in maintaining the integrity of the lung parenchyma when exposed to mechanical insult [4, 5]. Increasing evidence suggests that mechanical ventilation might lead to ventilator induced lung injury (VILI), even in healthy subjects [6]. Mechanical ventilation with imposed tidal volume (VT ) and positive end-expiratory pressure (PEEP) may promote primary damage to endothelial [7] and epithelial [8] cells, as well as to peripheral airways [9, 10] and to ECM macromolecules [11–14]. PGs seem to be particularly susceptible to changes in ventilatory settings: indeed, the GAGs component of the PGs molecules showed a VT -dependent fragmentation and washout [13] and increased deposition in the lung interstitium with excessively high VT [11]. The use of PEEP is generally considered protective when applied during mechanical ventilation, based on indications provided by experiments performed in animals with healthy [9, 15–17], and diseased [18] lungs. The beneficial effect of PEEP has been attributed to the fact that, by assuring a more homogenous lung expansion, PEEP prevents collapse and re-opening of alveolar spaces [12, 19, 20], an event which is more likely to occur in dependent regions of the lung. In fact, PEEP imposes greater strain on the alveolar structures, while probably reducing the intratidal changes in strain in open alveolar structures. However, in a previous study performed on healthy supine rats [12], we found that PEEP was indeed protective on lung parenchyma at low, but not at high VT where it contributed to increased alveolar pressure and lung expansion. An additional factor that may interfere with the impact of mechanical ventilation on lung parenchyma is fluid load, often delivered in the peri-operative phase and/or in the post-surgical clinical follow-up of the patients; indeed, in addition to reduction of the sieving properties of the capillary endothelium and of the alveolar barrier, plasma expansion may lead to a progressive loss of interstitial GAGs integrity [21–23]. In line with these observations, in a previous study performed analyzing the whole lung, we found that the simultaneous use of high VT , PEEP and fluid load exposes the lung parenchyma to an easier development of tissue edema [12]. However, in that study we neglected the fact that the mechanical stresses applied to the alveolar wall and to the pulmonary endothelium are not homogenously distributed throughout the lung and that, therefore, the combination of mechanical ventilation, PEEP and plasma expansion might non-homogenously

affect different lung regions. In the present study, we investigated possible differences induced by mechanical ventilation at low or high VT , with or without PEEP and with or without mild fluid load, on the morphology and the macromolecular assembly of the lung matrix in the ventral and dorsal lung regions of supine healthy rats. The results of this analysis might prove useful to establish whether posture dependent ventilation inequalities might expose specific areas of the lung parenchyma to a potentially greater hazard, thereby being particularly susceptible of developing lung injury during mechanical ventilation.

MATERIALS AND METHODS This study presents a further analysis of data partially published in a previous study [12] performed on anesthetized healthy rats. All experimental procedures and protocols were performed according to the “Principles of laboratory animal care” (NIH publication No. 86–23, revised 1985) and of the European Declaration of Animal Right and Welfare (2010/63/EU) and were approved by the Institutional Animal Care Committee at the University of Insubria, Varese, Italy in accordance with the Health Research Extension Act. Experiments were performed on 78 adult pathogen-free male Wistar rats (Charles River; Laboratories Italy, Calco, LC) (BW 290 ± 21.8 (SE) g) anaesthetized with an intraperitoneal injection of a cocktail composed by 75 mg/kg of ketamine (Imalgene 1000, Merial, Italy) and 0.5 mg·kg−1 of medetomidine (Domitor, Pfizer) in saline solution. The adequacy of anesthesia level was assessed on the basis of the disappearance of the noxious reflexes. Additional half-boluses of the same anaesthetic cocktail were administered every hour until the end of the experiments, when animals were euthanized via an anaesthesia overdose. Once deeply anaesthetized, the rats were turned supine on a heating pad set to maintain the body temperature at ∼37◦ C. After tracheotomy, a T-shaped cannula inserted into the trachea was connected to a heated Fleish pneumotachograph (model 8420, Hans Rudolph Inc.) equipped with a differential pressure transducer (2 cmH2 O; Validyne MP45, Northridge, CA) to continuously monitor airflow (V˙ E ). The flow signal was digitized with a National Instruments BNC-2090 analog-to-digital board (sampling rate: 100 Hz) and integrated by dedicated LabView software (National Instruments) to obtain respiratory tidal volume (VT ). Airways pressure (PAW ) was measured with a physiological pressure transducer (model P23XL; Gould Electronics) connected to the side arm of the tracheal cannula; there was no appreciable Experimental Lung Research

Lung Matrix in Mechanical Ventilation and Fluid Load

shift in the signal or alteration in amplitude up to 20 Hz. All recorded signals were amplified, sampled at 100 Hz by a 14-bit analog-to-digital converter and stored on a desk computer. Saline filled plastic catheters (PE50) were inserted into the carotid artery and the jugular vein to continuously monitor systemic arterial and central venous pressures. In rats exposed to extracellular fluid load, an additional catheter was inserted into a femoral vein to be connected to a continuous infusion pump (KDS 100 L, KD Scientific, Holliston, MA, USA).

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Experimental Protocols A first set of animals (not-infused, NI; n = 28, 4 rats per group) were randomly assigned to the following experimental groups: a) untreated controls, suppressed through an anaesthesia overdose without being exposed to any experimental instrumentation or treatment (group C0 , n = 4); b) anaesthetized and instrumented rats suppressed after 4 hours of spontaneous breathing (SB) with room air at 0 cmH2 O end-expiratory pressure (ZEEP) (group C4h ; n = 4); c) anaesthetized and instrumented rats suppressed after 4 hours of SB with room air at 5 cmH2 O PEEP (group C4h−P ; n = 4). In group C4h−P , PEEP was attained by adding an expiratory threshold realized by connecting the side port of the three ways tracheal cannula to a plastic catheter whose tip was immersed in a pool of saline at a depth setting the desired PEEP; d) anaesthetized and instrumented rats which, after 10 min baseline SB, were mechanically ventilated (ventilator model SPIRA, Harvard Instruments) for 4 hours with room air at ZEEP (group MVL ; n = 4) or PEEP-5 (group MVL−P ; n = 4) at the same VT and respiratory rate and therefore V˙ E as in baseline SB. Prior to the beginning of mechanical ventilation rats were paralyzed with a single bolus of 0.3 mL of pancuronium bromide (Sigma Aldrich, Milan, Italy) solution (2 mg/mL in saline) delivered in the femoral catheter; e) anaesthetized and instrumented rats which, after 10 min baseline spontaneous breathing, were mechanically ventilated for 4 hours with room air at ZEEP (group MVH ; n = 4) or PEEP5 (group MVH ; n = 4) maintaining the baseline V˙ E through a 3 fold higher VT compared to SB, and a correspondingly decreased (1/3 of SB) respiratory rate. A second set of animals (infused, I; n = 24) was used to verify whether an increased tissue hydration might have an additional impact, either protective or injurious, on lung parenchyma exposed to different ventilatory strategies. Therefore, rats ventilated as in previous points (b) through (e) in addition received an intravenous infusion of phosphate buffered saline  C

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(PBS) at a constant rate of 7 mL ·kg−1 ·hr−1 maintained for 4 hours. At the end of the period of either spontaneous or mechanical ventilation, the animals were suppressed with an anesthesia overdose. The lungs were immediately excised from the chest and rinsed in saline solution. After clearing from larger bronchi, samples of the lung parenchyma from the ventral and dorsal lung regions were immediately weighted to measure the wet tissue weight (W); they were then dried at 70◦ C in a speed vacuum apparatus for 24 h and weighted again to obtain the dry tissue weight (D). The wet weight to dry weight (W/D) lung ratio was then used as an index of extracellular extravascular lung water content. The remaining ventral and dorsal lung parenchyma was cut into smaller pieces and frozen in liquid nitrogen for subsequent biochemical analysis.

Biochemical Analysis The biochemical analysis consisted in the isolation, purification and characterization of GAG chains. The lyophilized lung samples were extracted with increasing concentration of guanidinium hydrochloride (GuHCl), 0.4 M followed by 4 M in 50 mM sodium acetate buffer, pH 5.6, containing protease inhibitors (Complete Mini EDTA-free tablets, Roche) at 4◦ C for 48 h [24]. Distinction between CS-GAGs, extracted from large PGs typical of the solid matrix structures, and HS-GAGs, extracted from PGs preferentially localized in the basal membrane of the endothelial and epithelial membranes, was performed by selective enzymatic digestion using respectively 100 mU/mL of Chondroitinase ABC (E.C. 4.2.2.4) and a mix of Heparinases I, II, III, (E.C.: I 4.2.2.7, II no number, III 4.2.2.8). The product of extraction was then analysed with the hydroxydiphenyl method of Blumenkrantz and Asboe-Hansen [25] as modified by van den Hoogen [26] who adjusted it for a 96 well microtitre plate. This modification is particularly useful for analyzing a large number of samples. Briefly, 40 μL of the extraction solution and 200 μL of concentrated H2 SO4 were added to each well of the plate. The plate was then incubated at 80◦ C for 60 min. After cooling the absorbance was read in a spectrophotometer at 540 nm. Subsequently, 40 μL of m-hydroxydiphenyl reagent (100 μL of 100 mg/mL mhydroxydiphenyl in dimethyl sulphoxide, mixed with 4.9 mL 80% sulphuric acid) were added, and after 15–30 min the absorbance at 540 nm was read again. Quantification of uronic acid was made against a glucuronic acid standard. The uronic acid content was expressed as μg of glucuronic acid mg−1 of dry tissue.

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Morphological Analysis In order to evaluate the impact of the ventilatory strategies on the lung tissue from the morphological standpoint, 26 additional rats were used. These animals (n = 2 per group) were randomly divided in 13 groups and were exposed to exactly the same surgical preparation and ventilatory procedures previously described for the not-infused (NI) and infused (I) rats. At the end of the experimental protocol, the rats were suppressed and the lung was fixed by intratracheal instillation with 4% Karnovsky’s solution, at a transpulmonary pressure of 20 cmH2 O [27]. Specimens from the ventral and dorsal lung regions were kept into the fixative solution for 6 h and then were washed in 0.1 M cacodylate buffer. The samples were postfixed in 1% OsO4 for 1 h, washed in 0.1 M cacodylate buffer, dehydrated in ascending ethanol and propylenoxyde, and then embedded in epon 812 mixture. The embedded specimens were cut with a RMC MTX ultramicrotome (Boeckeler Instruments Inc., Tucson, Arizona, USA) and the obtained semithin section were stained with Toluidin blue and observed with a light microscope (Nikon Eclipse E600). Quantitative morphological analysis of the ventral and dorsal lung parenchyma has been performed using an appropriate software for image acquisition and analysis (Simple PCI-Hamamatsu). On each acquired image the following parameters were measured: a) inter-alveolar septa thickness, b) alveolar radius; c) alveolar cross sectional area and d) area of the peripheral tissue region at alveoli confluence (corner region), obtained as the surface of the circle inscribed within the corner region. At the corners, the low radius of curvature of the adjacent alveoli reduces pulmonary interstitial fluid pressure; hence, corners represent potential site of fluid accumulation when interstitial edema develops.

Statistical Analysis Data are reported as mean ± SE. Comparison between values from different groups was performed by one–way ANOVA using pairwise multiple comparison procedures (Holm-Sidak method). Comparison between data obtained in different regions in the same animals was performed by paired Student’s t-test; while comparison between data obtained from the different strategies, was performed by unpaired Student’s t-test. Differences between mean values were considered significant for p < 0.05 and highly significant for p < 0.01.

RESULTS Mechanical ventilation setting and gas-exchange at different experimental protocols has been previously reported in detail in our previous study [12] and are summarized in Tables 1 and 2, respectively.

Regional Wet Weight to Dry Weight (W/D) Lung Ratio The regional lung W/D ratios measured at the end of the experiments in not-infused and infused groups are reported in Figure 1 A and B, respectively, distinguishing between the ventral (white bars) and the dorsal (black bars) lung regions. In not-infused MVL (Figure 1A), the W/D of the dorsal regions was lower compared to the corresponding MVL ventral lung regions and to the C4h values. In MVL–P , W/D was significantly increased in the ventral, while it was normal in the dorsal regions compared to C4h (Figure 1A). In MVH–P , the W/D ratio was significantly increased, compared to C4h−P , in all lung regions. In infused groups, the ventral to dorsal differences observed in not infused MVL and MVL–P disappeared and the W/D ratio significantly increased, compared to infused C4h and C4h−P , in MVH–P .

Morphological Analysis The analysis was performed on 915 images from notinfused and 715 images from infused lung sections, respectively. Histological sections from the ventral and dorsal regions of not-infused and infused lungs at the different ventilatory strategies are presented in Figure 2, panels A-N and Figure 3 panels A-N, respectively. In both not-infused and infused groups the parenchyma was well preserved with no sign of injury, although differences in alveolar septa thickness were observable in relation to ventilatory setting, lung region, and parenchyma hydration state. In not-infused groups a generalized thinning of the alveolar septa was observed in MVL (Figure 2 E, F), MVL−P (Figure 2 I, L), and in ventral region of MVH−P (Figure 2M), when compared to control C4h (Figure 2A). In addition, enlarged perivascular fluid cuffs were common in both the ventral and the dorsal lung (Figure 2M, N). In infused groups (Figure 3A-N), the parenchyma appeared slightly more congested than in corresponding not infused lungs, with patchy development of perivascular fluid cuffs around lung microvasculature. In all groups, positive PAW during mechanical ventilation induced alveolar septa thinning compared to infused-C4h (Figure 3A, B). The geometric means of alveolar septa thickness and of Experimental Lung Research

Lung Matrix in Mechanical Ventilation and Fluid Load TABLE 1.

Respiratory Parameters in Spontaneous Breathing and Mechanical Ventilation Spontaneous breathing

NOT INFUSED VT mL RF breath/min V˙ E mL /min PPEAK−AW cmH2 O INFUSED VT mL RF breath/min V˙ E mL /min

Mechanical ventilation

C4h

C4h−P

MVL

MVH

MVL−P

MVH−P

2.2 ± 0.2 46.7 ± 1.3 100.7 ± 4.7

1.3 ± 0.2 24.6 ± 1.2∗ 48.4 ± 4.9∗ 5 ± 0.5

2.9 ± 0.3 33.7 ± 1.2 86.1 ± 4.6 16.2 ± 0.7‡

7.5 ± 0.6 12.7 ± 1.4 93.7 ± 10.1 22.5 ± 1.2

2.8 ± 0.2 41 ± 9.6 110.5 ± 15.8 18.8 ± 0.7

8 ± 0.5 13.3 ± 2.0 105.1 ± 12.2 32.8 ± 1.4‡

I-C4h 2.7 ± 0.2 46 ± 8.2 122.2 ± 23.1

I-C4h−P 2.8 ± 0.05 33.5 ± 2.2 95.8 ± 8.2

I-MVL 2.7 ± 0.3 45 ± 6.7 118.2 ± 11.5

I-MVH 7.9 ± 0.7 13.2 ± 2.2 107.7 ± 24.9

I-MVL−P 2.9 ± 0.05 43.5 ± 0.5 126.2 ± 3.5

I-MVH−P 8.5 ± 0.4 13 ± 1.7 104.4 ± 14.7

19.7 ± 0.2

40.2 ± 5.2

0 ± 0.5

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5 ± 0.5

16.7 ± 1.0

31.1 ± 3.0§

Average respiratory parameters in spontaneously breathing or mechanically ventilated not infused (C4h and C4h−P ; MVL , MVH , MVL−P , and MVH−P ) and infused (I-C4h and I-C4h−P ; I-MVL , I-MVH , I-MVL−P , and I-MVH−P ) rats (n = 4 per group). VT : tidal volume; RF: respiratory frequency; V˙ E , minute ventilation. Ppeak-aw, peak airways pressure. Values are mean ± SE. ∗ Significantly different (P < .05) from C4h . ‡ Significantly different (P < .05) from corresponding baseline value. § significantly different (P < .05) from corresponding value in not-infused group.

extra-alveolar extracellular tissue area at alveolar confluence (alveolar corners) are presented in Figures 4 and 5, respectively, for the not-infused (upper panels) and infused (lower panels) groups distinguishing between the ventral (white bars) and the dorsal (black bars) regions. In not-infused C4h (Figure 4A), alveolar septa thickness was 37.2% lower in the dorsal than in the ventral lung. In the ventral regions, mechanical ventilation at positive PAW was associated with an alveolar septa thickness reduction, with respect to C4h , of - 39.5% in C4h−P , - 58% in MVL , -

TABLE 2.

64.1% in MVL−P , −59.9% in MVH−P and −31.3% in MVH ; alveolar septa thinning was less pronounced in the dorsal MVL (−17.9% of C4h ), MVH (−14,0%) and MVH−P (−31.9%) regions as compared to corresponding ventral ones. Infused groups showed, with respect to corresponding not-infused ones, a significant thickening of interalveolar septa. In addition, the ventral-to-dorsal differences in septa thickness were reduced in infused compared to not-infused groups (Figure 4). In notinfused groups extra-alveolar corner area (Figure 5 A)

Hemogas Analysis after 4 hours of Spontaneous or Mechanical Ventilation Spontaneous breathing

Not Infused

C4h

Mechanical ventilation

C4h−P

MVL ∗∗

Pa O2 mmHg Pa CO2 mmHg pHa INFUSED

83.7 ± 1.5 54.9 ± 2.7 7.3 ± 0.03 I-C4h

120.3 ± 3.5 46.2 ± 2.2 7.3 ± 0.03 I-C4h−P

Pa O2 mmHg Pa CO2 mmHg

92.1 ± 9.9 52.2 ± 2.4

90.0 ± 4.2‡ 52.9 ± 2.8

pHa

7.3 ± 0.01

7.3 ± 0.01

MVH ∗

50.7 ± 0.3 81.4 ± 7.5∗ 7.1 ± 0.03∗ I-MVL

44.8 ± 1.5∗§†† 83.8 ± 3.1∗

7.0 ± 0.02∗∗§

MVL−P

MVH−P

75.3 ± 3.9 56.9 ± 2.3 7.3 ± 0.01 I-MVH

100.7 ± 11.2 76.3 ± 7.1∗ 7.2 ± 0.03∗ I-MVL−P

130.7 ± 6.8∗ 45.1 ± 4.5 7.2 ± 0.04 I-MVH−P

81.1 ± 3.2 63.8 ± 6.0

91.3 ± 8.2 67.5 ± 3.5∗

142.6 ± 8.5∗ 53.9 ± 3.0

7.1 ± 0.02∗§

††

7.1 ± 0.02∗§

7.2 ± 0.02

∗Significantly different (P < .05) from C4h . ∗∗ Significantly different (P < .01) from C4h . †† Significantly different (P < .05) from MVL § Significantly different (P < .05) from corresponding not infused. ‡ Significantly different (P < .01) from corresponding not infused. Arterial blood gas analysis at the end of the experiment in spontaneously breathing or mechanically ventilated not infused (C4h and C4h−P ; MVL , MVH , MVL−P , and MVH−P ) and infused (I-C4h and I-C4h−P ; I-MVL , I-MVH , I-MVL−P and I-MVH−P ) rats (n = 4 per group). Values are mean ± SE. pHa , arterial pH; Pa CO2 , arterial PCO2 ; Pa O2 , arterial PO2 .  C

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FIGURE 1. Wet weight to dry weight ratio (W/D) of tissue samples excised from the ventral (white bars) or from the dorsal (black bars) lung regions at the end of the 4-h experiment in not-infused (panel A) and infused (panel B) rats. Bars represent mean ± SE. ∗ (P < .05), ∗∗ (P < .01);

ventral versus dorsal, § (P < .05), §§ (P < .01); not-infused versus infused, # (P < .05), versus C4h, & (P < .05), versus C4h-P.

behaved similarly to alveolar septa thickness with similar differences among groups and, within each group, between ventral and dorsal areas. In infused groups, fluids caused a generalized increase of extra-alveolar corner area in all samples but in the ventral regions of infused MVL and MVH . As observed also for septa thickness, regional differences in extra-alveolar corner area were reduced in infused compared to notinfused lungs (Figure 5).

Biochemical Analysis The amount of CS/DS-GAGs and HE/HS-GAGs extracted from the ventral and dorsal regions of notinfused and infused lungs is presented in Figure 6. In not-infused groups, MVL caused a marked drop (−89.2% in the ventral and −73.1% in the dorsal regions) of CS/DS-GAGs extraction (Figure 6 A), with no significant impact on HE/HS-GAGs

FIGURE 2. Toluidine blue stained hystological sections of the ventral and dorsal lung parenchyma of not-infused rats after 4 hours of spontaneous breathing (C4h , panels A, B; C4h−P , panels C, D) or mechanical ventilation (MVL , panels E, F; MVH , panels G, H; MVL−P , panels I, L; MVH−P , panels M, N). Scale bars = 100 μm.

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FIGURE 4. Average thickness of the interalveolar septa measured on images of the ventral (white bars) or dorsal (black bars) lung regions such as shown in Figures 2 and 3, in lung samples from not-infused (panel A) and infused (panel B) rats. N = 4 per group. Bars represent mean ± SE. ∗ (P < .05) ∗∗ (P < .01); ventral versus dorsal. § not-infused versus infused. # vs C4h, & vs C4h-P.

FIGURE 3. Toluidine blue stained hystological sections of the ventral and dorsal lung parenchyma of infused rats after 4 hours of spontaneous breathing (C4h , panels A, B; C4h−P , panels C, D) or mechanical ventilation (MVL , panels E, F; MVH , panels G, H; MVL−P , panels I, L; MVH−P , panels M, N). Interstitial fluid cuffs (arrows) were often observed around lung microvasculature with no sign of alveolar edema. Scale bars = 100 μm.

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(Figure 6 B). A similar, although milder, effect was observed in MVH , which showed a significant decrease of CS/DS- GAGs extraction in the dorsal region. Application of PEEP in C4h−P caused a mild but significant drop of CS/DS-GAGs compared to C4h (Figure 7 A) in the dorsal region, with no effect on HE/HS GAGs (Figure 7 B). Combining PEEP with mechanical ventilation in not-infused groups did not further affect the CS/DS-GAGs, but decreased HE/HS-GAGs extraction in the ventral regions of MVL−P and MVH−P . CS/DS and HE/HS-GAGs in the dorsal regions of C4h (Figure 6 C,D) were lower in infused as compared to not-infused groups. Mechanical ventilation increased both CS/DS- and HE/HSGAGs extraction, particularly in the ventral regions and more markedly in MVH than in MVL groups. Application of PEEP increased, with respect to corresponding not-infused groups, the CS/DS-GAGs extraction in the dorsal regions of C4h−P and MVH−P

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FIGURE 5. Extra-alveolar corner area measured on images of the ventral (white bars) or from the dorsal (black bars) lung regions from images such as shown in Figures 2 and 3, in lung samples from not-infused (panel A) and infused (panel B) rats. N = 4 per group. Bars represent mean ± SE. ∗ (P < .05) ∗∗ (P < .01); ventral versus dorsal, § not-infused versus infused, # vs C4h, & vs C4h-P.

(Figure 7 C) and in the ventral region of MVL−P . HE/HS-GAGs extraction also increased with PEEP, in particular in the ventral regions of infused-MVH−P (Figure 7 D).

DISCUSSION The results of the present analysis indicate that the impact of mechanical ventilation on supine healthy rat lungs is not homogenous throughout the parenchyma and is affected by the VT size: low VT altered both the ventral and dorsal lung regions, whereas high VT selectively injured the dorsal regions. PEEP was more effective in minimizing lung injury in the ventral compared to dorsal lung regions independent of VT value. Intravascular fluid load extended the lesional effect of mechanical ventilation, particularly in the ventral lung. Hence, our data suggest that during controlled mechanical ventilation the ventral

FIGURE 6. GAGs extraction in control and mechanically ventilated lungs at 0 cmH2 O end-expiratory pressure (ZEEP). A, B: CS/DS- (panel A) and HE/HS-GAGs (panel B) extraction from the ventral (white bars) or from the dorsal (black bars) lung regions in controls (C4h ) and in lungs mechanically ventilated at low VT (MVL ) or high VT (MVH ) at 0 cmH2 O end-expiratory pressure (ZEEP) in not-infused rats. C, D: CS/DS- (panel C) and HE/HS-GAGs (panel D) extraction from the ventral (white bars) or from the dorsal (black bars) lung regions in infused controls (C4h ) and in infused lungs mechanically ventilated at low VT (MVL ) or high VT (MVH ) at 0 cmH2 O end-expiratory pressure (ZEEP) in infused rats. Bars represent mean ± SE. N = 4 in each group.∗ (p < .05) ∗∗ (p < .01); ventral versus dorsal. § not-infused versus infused. # vs C4h, & vs C4h-P.

regions are more susceptible than the dorsal ones to the mechanical and hydrodynamic stresses.

Adequacy of the Present Rat Model We performed the experiments in supine rather than prone animals for two main reasons: a) to compare the present data to those obtained from previous studies performed in supine rats with similar ventilatory strategies [12, 13] and b) to take advantage of the fact that greater lung regional differences have been reported in the supine compared to the prone position [28, 29]. It is worth noting, however, that modifications in ventilation and/or perfusion when changing Experimental Lung Research

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tion might not instantaneously equilibrate. In fact, an increase in interstitial fluid content depends upon:

FIGURE 7. GAGs extraction in controls and spontaneously breathing or mechanically ventilated lungs expanded at 5 cmH2 O positive end-expiratory pressure (PEEP) A, B: CS/DS- (panel A) and HE/HS-GAGs (panel B) extraction from the ventral (white bars) or from the dorsal (black bars) lung regions in controls at ZEEP (C4h , from Figure 6), controls at 5 cmH2 O end-expiratory pressure PEEP (C4h−P ) and in lungs mechanically ventilated with a low VT (MVL−P ) or high VT (MVH−P ) at PEEP in not infused rats. C, D: CS/DS- (panel C) and HE/HS-GAGs (panel D) extraction from the ventral (white bars) or from the dorsal (black bars) lung regions in controls at ZEEP (C4h , from Figure 6), controls at 5 cmH2 O end-expiratory pressure PEEP (C4h−P ) and in lungs mechanically ventilated with a low VT (MVL−P ) or high VT (MVH−P ) at PEEP in infused rats. Bars represent mean ± SE. N = 4 in each group. ∗ (P < .05) ∗∗ (P < .01); ventral versus dorsal. § not-infused versus infused, # vs C4h, & vs C4h-P.

posture may be at least partially masked by the fact that parenchyma density is always higher in the most dependent lung regions [30]. Septa thickness and alveolar corner area measurements in the present experiments include the contribution of both pulmonary vasculature and surrounding interstitium. Turning the animal from its normal prone to supine posture increases pulmonary intravascular pressure, blood perfusion and possibly tissue hydration in the dorsal compared to the ventral regions. While intravascular pressure and perfusion change at once when turning posture, tissue hydra C

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1. an increased trans-endothelial pressure gradient driving fluid from the vascular to the extravascular compartment. Intravascular fluid pressure changes by 1 cmH2 O/cm lung height, while pulmonary interstitial pressure (Pi ) decreases by ∼ 0.8 cmH2 O/cm [31]. Hence, for a rat lung height of ∼ 3cm, the net trans-endothelial hydraulic pressure gradient in a certain lung region ought to be only slightly modified when changing posture; 2. an increased endothelial hydraulic conductivity. However, our data (Figure 6) show that HS/HEGAGs extraction from C4h lungs is similar in the ventral and dorsal lung regions. Since HS/HEPGs are typical components of vascular basement membranes and contribute to their sieving properties [32] this suggests that the permeability of the capillary endothelium is homogeneous throughout the lung. 3. the occurrence of a ventral to dorsal fluid flux. The existence of a vertical pressure gradient of ∼ 0.8 cmH2 O/cm [31] in the lung parenchyma yields a potential gravity dependent fluid flux in the lung interstitium, similarly to what observed in the pleural space. However, at variance with what occurs in the matrix-free pleural cavity, pulmonary fluid slowly moves within the porosity of the extracellular solid matrix. It is worth noting that these experiments were performed on healthy lungs whose interstitial fluid volume is minimised, as documented by the low W/D ratio of the C4h lungs (Figure 1). The low interstitial mechanical compliance [21] may well contribute to reduce the fluid flow velocity within the matrix which is ∼ 0.1÷ 0.6 μL/sec in the interstitium of the rodent ear window [33]. Hence, interstitial fluid shift might take longer than the experimental time (4 h) to completely redistribute from the ventral (previously dorsal in the prone position) regions; 4. the local lymphatic drainage, which, as a consequence of the higher Pi values [31], is likely enhanced in the dorsal parenchyma. Because of these factors, pulmonary interstitial fluid volume would not immediately increase in the dorsal regions when turning from prone to supine posture. Indeed lung morphology (Figures 2 and 3) and quantitative analysis (Figures 4 and 5) indicate that in spontaneous ventilation (C4h ) septa thickness and alveolar corner area remained larger in the ventral than in the dorsal lung regions, still reflecting the normal prone posture distribution. On the other hand, in infused-C4h lungs, septa thickness and alveolar corner

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area increased more in dorsal than in ventral regions, suggesting that, in supine posture, vascular perfusion and perivascular tissue hydration tend to be higher in the dorsal lung. Regional GAGs extraction in C4h and infused-C4h lungs is in line with these considerations. Indeed, while in normally hydrated C4h lungs the regional CS/DS- and the HE/HS-GAGs content is rather uniform (Figure 6 A, B), in infused-C4h lungs both CS/DS- and HE/HS-GAGs extraction is significantly decreased (Figure 6 C, D) in the dorsal parenchyma, suggesting that a greater increase in vascular perfusion caused a greater degradation and GAGs washout in the dorsal lung regions.

Regional Impact of Mechanical Ventilation at ZEEP or PEEP In not-infused lungs, mechanical ventilation at low VT caused CS/DS-GAGs degradation and washout (Figure 6 A) throughout the whole lung, but particularly in the ventral regions; at high VT the lesional effect was mainly localized in the dorsal region, in line with what previously found in rodents [20, 34]. Since minute ventilation was kept constant among various experimental groups, ventilation at low VT required a higher respiratory rate and viceversa. The greater CS/DS-GAGs degradation with ventilation at low VT may thus reflect a combination of the mechanical impact of the frequent expansion/collapse cycles of lung parenchyma and increased local tissue strain/stress [12]. Since in supine posture at end-expiration the alveoli are more distended in the ventral than in the dorsal region, the imposed mechanical strain might exert a more lesional overdistending effect in the former [35]. A modified matrix architecture and therefore a more fragile parenchyma might explain the significant thinning of interaveolar septa observed in ventral regions in MVL lungs (Figure 4 A). Although fragile, the alveolar parenchyma was not severely damaged, in line with results showing that even injurious ventilation does not determine a direct lesion of the alveolar wall [36]. It is worth noting that GAGs degradation is usually considered as a sign of disorganization of the matrix which tends to become more compliant and to accommodate more fluid [12], favoring the development of local interstitial edema. However, in the present study, the CS/DS- degradation in the ventral lung coexists with a normal HE/HS-GAGs extraction (Figure 6 B) suggesting that no fluid accumulation occurs (Figure 1) provided the endothelial barrier integrity is preserved. The fact that with low frequency mechanical ventilation at high VT (MVH ) CS/DS-GAGs degradation was markedly reduced and present only in the dorsal lung (Figure 6A), suggests that the most damaging

factor on structural matrix macromolecules is not the positive peak alveolar pressure (see Table 1), higher in MVH than in MVL , but rather the combination of imposed alveolar expansion/collapse and higher respiratory rate. In addition, mechanical ventilation at low VT seems to be more injurious on the ventral, likely less ventilated alveoli, where Pa CO2 and Pa O2 are expected to be respectively higher and lower than in the dorsal areas, leading to local acidosis and tissue sufferance, both factors being potential causes of tissue damage [12]. The use of PEEP, by limiting the collapse and reopening of alveolar spaces and more homogenously expanding the alveoli and the small airways [37], reduces the impact of mechanical ventilation [12, 19, 20]. Interestingly, PEEP eliminates the differences of alveolar septal thickness and corner area observed between dorsal and ventral regions in C4h lungs, suggesting a more homogenous distribution of freely moving interstitial fluid within the parenchyma. PEEP was clearly protective in MVL−P , particularly in the ventral region parenchyma, where CS/DS-GAGs extraction was not altered compared to C4h (Figure 7A). The mild CS/DS-GAGs disorganization of the dorsal parenchyma of C4h−P suggests that PEEP is more protective on the less compliant ventral alveoli. The protective effect of PEEP did not extend to HE/HS GAGs which were considerably fragmented and washed out both at the ventral and at the dorsal regions of MVH−P lungs. Hence, although notinfused lungs were not clearly edematous, in MVH−P the W/D lung ratio was significantly higher compared to control C4h lungs, suggesting that either the endothelial permeability to water or the local tissue compliance or both were increased in lungs ventilated with high volumes and PEEP. The present results partially differ from previous ones [20, 38] showing that, at ZEEP, high VT induces greater injury, particularly in the dorsal regions, and that PEEP is protective at high VT . The diversity of these conclusions might depend upon the experimental model used: indeed, in the present experiments, targeted at studying the response of interstitial molecules to mechanical and hydrodynamic stresses, the W/D ratio was still normal or only slightly elevated, index of a mild interstitial, with no alveolar edema, endothelial or epithelial lesion. Other studies, aimed instead at finalizing the most protective and more efficient strategy to ventilate injured lungs, were necessarily performed on lungs with a high W/D ratio, significant alveolar flooding, epithelial and endothelial injury and inflammatory response [38]. Hence, the apparently controversial response of regional lung parenchyma to VT and PEEP might reflect the degree of interstitial injury, which was limited in our Experimental Lung Research

Lung Matrix in Mechanical Ventilation and Fluid Load

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experiments, and the unequal distribution of edematous alveolar fluid, which was not encountered in our model. Our previous investigation [12] showed that, at least for up to 4 hours, mechanical ventilation, with or without fluid load, did not trigger an inflammatory response in the lung tissue. However, one cannot completely rule out the possibility that the regional extracellular tissue alterations observed in lungs exposed to the various mechanical ventilation strategies might be attributable to a local yet undetected inflammatory process.

Regional Impact of Mechanical Ventilation Associated to Fluid Load The W/D ratio is a non specific index of interstitial and/or alveolar edema progression, when it increases above the normal value; the lowest W/D ratio in not infused MVL lungs can be interpreted as a reduction, compatible with the low CS/DS-GAG extraction in this group, of the hydrophilic macromolecules in the tissue, thus shifting the equilibrium between free moving water and chemically bound water toward a lower than normal tissue water content. The lung W/D ratio was only slightly higher in infused compared to not-infused rats, indicating the development of a mild interstitial, but not alveolar, edema. Fluid accumulation affected the whole lung, as witnessed by the rather homogeneous increased of septa thickness and corner area, with disappearance of the dorso–ventral difference observed in C4h lungs. With respect to not-infused lungs, fluid load increased the extraction of both CS/DS- and HE/HS-GAGs in most ventilatory strategies. Since the present GAGs analysis excluded contamination by hyaluronan and allowed a complete extraction of GAGs from the tissue specimen, the observed increase in GAGs extraction might only reflect a contamination of GAG fragments mobilized from extrapulmonay tissues and transported to the lung through the pulmonary circulation. Indeed, the greater increase of HE/HS-GAGs compared to CS/DS-GAGs extraction suggests that fluid load, by increasing tissue perfusion and likely shear stress at the vessels wall [39], might trigger a faster endothelial basement membrane remodeling, particularly in the dorsal parenchyma, where blood flow ought to be higher than in the ventral one. Instead, the most remarkable CS/DS- and HE/HS-GAGs extraction was observed in the ventral zones of spontaneously breathing C4h and mechanically ventilated MVL and MVH infused lungs ventilated at ZEEP, suggesting that increased plasma volume and cardiac output tend to preferentially recruit previously poorly perfused lung regions rather  C

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than following a preferential gravity-dependent distribution to the dorsal lung parenchyma [19]. Interestingly, PEEP was accompanied by a greater fragmentation of CS/DS-GAGs in the ventral parenchyma of infused-MVL−P and of HE/HS-GAGs in the ventral parenchyma of infused-MVH−P, suggesting again that ventral regions are more vulnerable than the dorsal ones towards mechanical and hydrodynamic tissue stresses. In addition, one cannot exclude that the increased GAGs extraction observed in the ventral lung might reflect local deposition of new GAGs synthesized from a pool of tissue proteins available for fast PG synthesis in response to strain-induced matrix breakdown in the lung tissue [40–42]. Indeed, a significant increase of the protein component of CSPG and HS-PGs and a strengthening of the GAGs bonds has been reported after 2 hours of mechanical ventilation at high VT [11].

Study Limitations Our experimental protocol presents the following limitations: (a) it was performed on healthy rats, so that results cannot be directly extrapolated to larger animals or to experimental models of lung injury; (b) in rats, the injurious regional effects of mechanical ventilation might have been underestimated due to the reduced transpulmonary ventral- to-dorsal gradient; (c) the biochemical analysis was limited to PGs ignoring the regional distribution of other interstitial fibrillar components or inflammatory mediators in the lung tissue and in the blood; (d) the results obtained by infusing cristalloids might change with other types of fluids; (e) the number of animals used for morphological analysis was limited, a choice based on the fact that, in each animal, an extremely high number of images and lung structures were analysed. However, we found no differences in the indications provided by the two lungs, suggesting that the present morphological analysis may actually provide useful hints in the interpretation of the biochemical and functional data.

Conclusions In healthy rats, mechanical ventilation and fluid load may cause additive injuries to the lung parenchyma. Indeed, mechanical stresses, associated to low VT without PEEP prevalently affect dorsal lung regions, while increased fluid load likely induces a shear stressdependent injury in the ventral lung regions. Application of PEEP, at low VT ventilation might reduce ECM fragmentation and lung injury in both ventral and dorsal lung regions. Our data suggest that extreme caution is required in choosing the appropriate

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combination of ventilatory setting and fluid load even when ventilating normal lungs.

ACKNOWLEDGMENTS

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The authors gratefully acknowledge the “Centro Grandi Attrezzature per la Ricerca Biomedica” of Insubria University for instruments availability. Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. The authors alone are responsible for the content and writing of the article. This research was funded by a Research Award of the European Society of Anesthesiology (ESA 2007) and by the University of Insubria Research funding (FAR).

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